CN110573522B - SIRP alpha-41 BBL fusion proteins and methods of use thereof - Google Patents

SIRP alpha-41 BBL fusion proteins and methods of use thereof Download PDF

Info

Publication number
CN110573522B
CN110573522B CN201880015833.1A CN201880015833A CN110573522B CN 110573522 B CN110573522 B CN 110573522B CN 201880015833 A CN201880015833 A CN 201880015833A CN 110573522 B CN110573522 B CN 110573522B
Authority
CN
China
Prior art keywords
cells
bbl
fusion protein
cancer
sirpa
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201880015833.1A
Other languages
Chinese (zh)
Other versions
CN110573522A (en
Inventor
诺姆·沙尼
约西·戈兹兰
米哈尔·杰奈斯基艾尔哈里欧
埃德温·布莱德
伊都·卡明斯基
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kahr Medical Ltd
Original Assignee
Kahr Medical Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kahr Medical Ltd filed Critical Kahr Medical Ltd
Publication of CN110573522A publication Critical patent/CN110573522A/en
Application granted granted Critical
Publication of CN110573522B publication Critical patent/CN110573522B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2893—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD52
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Abstract

The invention provides a SIRP alpha-41 BBL fusion protein. Thus, a sirpa-41 BBL fusion protein is provided that comprises a single amino acid linking chain between sirpa and 41 BBL. The invention also provides a SIRPalpha-41 BBL fusion protein in at least a homotrimeric form. The invention also provides polynucleotides and nucleic acid constructs encoding the SIRPalpha-41 BBL fusion proteins, host cells expressing the SIRPalpha-41 BBL fusion proteins, and methods of using the SIRPalpha-41 BBL fusion proteins.

Description

SIRP alpha-41 BBL fusion proteins and methods of use thereof
Background
Dual Signaling Proteins (DSPs), also known in the art as signaling proteins (SCPs), are currently known in the art as bifunctional fusion proteins that link an extracellular portion (extracellular amino-terminus) of a first type of membrane protein to an extracellular portion (extracellular carboxy-terminus) of a second type of membrane protein to form a fusion protein having two active sides (see, e.g., U.S. patent publication No. 7,569,663 and U.S. patent publication No. 8,039,437, both of which are incorporated herein by reference as if fully set forth herein).
Sirpa (signal regulatory protein α) is a cell surface receptor of an immunoglobulin superfamily. Sirpa is predominantly expressed on the surface of immune cells from the phagocytic lineage, such as macrophages and Dendritic Cells (DCs). CD47 is a ligand for sirpa. CD47 is a cell surface molecule in the immunoglobulin superfamily. CD47 functions as an inhibitor of phagocytosis by linking to sirpa that is displayed on multiple phagocytes. CD47 is widely expressed on most normal tissues. In this way, CD47 acts as a "snatch my signal" and an autologous marker, as the loss of CD47 results in constant state phagocytosis of aged or damaged cells. CD47 has been found to manifest on a variety of human tumor types. Tumors escape phagocytosis by macrophages through a variety of anti-phagocytosis signals, including CD 47. While CD47 is commonly expressed to a low degree on normal cells, many tumors exhibit a higher degree of CD47 relative to their normal cellular counterparts, and excessive expression of CD47 enables the various tumors to evade surveillance of the innate immune system by escaping phagocytosis.
4-1BBL is an activating ligand for the 41BB receptor (CD 137), a member of the TNF receptor superfamily, and a costimulatory molecule effective to induce T cell activation. 41BBL naturally forms a homotrimer, but efficient oligomerization of 4-1BBL is required for signaling through 4-1 BB. 4-1BBL is presented on a variety of Antigen Presenting Cells (APCs), including Dendritic Cells (DCs), B cells, and macrophages. The 4-1BB receptor was not detected (< 3%) on resting T cells and T cell lines, however, 4-1BB was stably upregulated when multiple T cells were activated. Activation of 4-1BB upregulates multiple surviving genes, enhances cell differentiation, induces cytokine production, and prevents activation-induced cell death in T cells.
Additional background art includes:
international patent application publication No. WO2017/059168;
international patent application publication No. WO2001/049318;
international patent application publication No. WO2016/139668;
international patent application publication No. WO2014/106839;
international patent application publication No. WO2012/042480;
U.S. patent application publication No. 20150183881;
U.S. patent application publication No. US 20070110746;
U.S. patent application publication No. US 20070036783; a kind of electronic device with high-pressure air-conditioning system
U.S. patent publication No. US 9562087.
Disclosure of Invention
According to an aspect of some embodiments of the invention, there is provided a sirpa-41 BBL fusion protein comprising a single amino acid linking chain between sirpa and 41 BBL.
According to an aspect of some embodiments of the invention, there is provided a sirpa-41 BBL fusion protein in the form of at least one homotrimer.
According to some embodiments of the invention, the at least one homotrimer has a molecular weight of at least 140 kilodaltons as determined by SDS-PAGE electrophoresis.
According to some embodiments of the invention, the sirpa-41 BBL fusion protein comprises a connecting strand between the sirpa and the 41 BBL.
According to some embodiments of the invention, the linking chain has a length of 1 to 6 amino acids.
According to some embodiments of the invention, the linking chain is a single amino acid linking chain.
According to some embodiments of the invention, the linking chain is not a crystallizable fragment domain of an antibody or a fragment of the crystallizable fragment domain.
According to some embodiments of the invention, the linking chain is glycine.
According to some embodiments of the invention, the sirpa-41 BBL fusion protein is soluble.
According to some embodiments of the invention, the sirpa comprises an extracellular domain of the sirpa or a functional fragment of the extracellular domain.
According to some embodiments of the invention, the 41BBL comprises an extracellular domain of the 41BBL or a functional fragment of the extracellular domain.
According to some embodiments of the invention, the sirpa-41 BBL fusion protein is capable of at least one of:
(i) Binding CD47 and 41BB;
(ii) Activating a message delivery pathway of the 41BB in a cell exhibiting the 41BB;
(iii) Co-stimulating a plurality of immune cells expressing the 41BB; and/or
(iv) Phagocytosis of a plurality of pathological cells expressing the CD47 by a plurality of phagocytes is enhanced compared to in the absence of the sirpa-41 BBL fusion protein.
According to some embodiments of the invention, the amino acid sequence of the SIRPalpha-41 BBL fusion protein comprises SEQ ID NO. 1.
According to some embodiments of the invention, the amino acid sequence of the SIRPalpha-41 BBL fusion protein consists of SEQ ID NO. 1.
According to some embodiments of the invention there is provided a polynucleotide encoding the sirpa-41 BBL fusion protein of the invention.
According to some embodiments of the invention, there is provided a nucleic acid construct comprising the polynucleotide of the invention, and a regulatory element for directing expression of the polynucleotide in a host cell.
According to some embodiments of the invention, the polynucleotide comprises SEQ ID NO. 8.
According to some embodiments of the invention, there is provided a host cell comprising the sirpa-41 BBL fusion protein of the invention, or the polynucleotide or the nucleic acid construct of the invention.
According to some embodiments of the invention, there is provided a method for making a sirpa-41 BBL fusion protein, the method comprising: the polynucleotides or the nucleic acid constructs of the invention are expressed in a host cell.
According to some embodiments of the invention, the method comprises: isolating the fusion protein.
According to some embodiments of the invention, the cell is selected from the group consisting of chinese hamster ovary Cells (CHO), perc.6 cells, and human embryonic kidney cells (293).
According to some embodiments of the present invention there is provided a method for treating cancer, the method comprising: the SIRPalpha-41 BBL fusion proteins of the invention are administered to a subject in need thereof.
According to some embodiments of the present invention there is provided a method for treating a disease that may benefit from activating a plurality of immune cells, the method comprising: administering the sirpa-41 BBL fusion protein of the invention, the polynucleotide of the invention, or the nucleic acid construct of the invention, or any one of the host cells of the invention, to a subject in need thereof.
According to an aspect of some embodiments of the present invention there is provided an article of manufacture identified for treating a disease that may benefit from activating a plurality of immune cells, the article of manufacture comprising: a packaging material containing a therapeutic agent for treating the disease; and a SIRPalpha-41 BBL fusion protein, a polynucleotide encoding the fusion protein, a nucleic acid construct encoding the fusion protein, or a host cell expressing the fusion protein.
According to some embodiments of the invention, the disease comprises a hyperproliferative disease.
According to some embodiments of the invention, the hyperproliferative disease comprises sclerosing or fibrosis, idiopathic pulmonary fibrosis, psoriasis, systemic sclerosis/scleroderma, primary cholangitis, primary sclerosing cholangitis, liver fibrosis, prevention of radiation-induced pulmonary fibrosis, myelofibrosis, or retroperitoneal fibrosis.
According to some embodiments of the invention, the hyperproliferative disease comprises cancer.
According to an aspect of some embodiments of the present invention there is provided a method for treating cancer, the method comprising: an anticancer agent; and a SIRPalpha-41 BBL fusion protein, a polynucleotide encoding the fusion protein, a nucleic acid construct encoding the fusion protein or a host cell expressing the fusion protein, to a subject in need thereof.
According to some embodiments of the invention, the cancer is selected from the group consisting of lymphoma, leukemia, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, and squamous cell carcinoma.
According to some embodiments of the invention, the plurality of cells of the cancer exhibit CD47.
According to some embodiments of the invention, the disease comprises a disease associated with immunosuppression or drug-induced immunosuppression.
According to some embodiments of the invention, the disease comprises Human Immunodeficiency Virus (HIV), measles, influenza, lymphocytic choriomeningitis (LCCM), respiratory fusion virus (RSV), human rhinovirus, human herpesvirus (EBV), cytomegalovirus (CMV), or a picovirus.
According to some embodiments of the invention, the disease comprises an infection.
According to some embodiments of the invention, the plurality of diseased cells of the subject exhibit CD47.
According to an aspect of some embodiments of the present invention there is provided a method for activating a plurality of T cells, the method comprising: activating a plurality of T cells in vitro in the presence of a sirpa-41 BBL fusion protein and a plurality of cells exhibiting CD47.
According to an aspect of some embodiments of the present invention there is provided a method for activating a plurality of phagocytes, the method comprising: activating a plurality of phagocytes in vitro in the presence of a sirpa-41 BBL fusion protein and a plurality of cells exhibiting CD47.
According to an aspect of some embodiments of the present invention there is provided a method for activating a plurality of immune cells, the method comprising: activating the plurality of immune cells in vitro in the presence of a sirpa-41 BBL fusion protein, a polynucleotide encoding the fusion protein, a nucleic acid construct encoding the fusion protein, or a host cell expressing the fusion protein.
According to some embodiments of the invention, the activating is performed in the presence of a plurality of cells exhibiting CD47 or exogenous CD47.
According to some embodiments of the invention, the plurality of cells exhibiting the CD47 comprises a plurality of pathological cells.
According to some embodiments of the invention, the plurality of pathological cells comprises a plurality of cancer cells.
According to some embodiments of the invention, the cancer is selected from the group consisting of lymphoma, carcinoma, and leukemia.
According to some embodiments of the invention, the activating is performed in the presence of an anti-cancer agent.
According to some embodiments of the invention, the anti-cancer agent comprises an antibody.
According to some embodiments of the invention, the antibody is selected from the group consisting of rituximab, cetuximab, trastuzumab, ibritumomab, alemtuzumab, gemtuzumab, ibritumomab, panitumomab, belimumab, bevacizumab, bivalizumab-maytansine, borrelimumab, brucemide, rituximab-vitamin, cetuximab, daclizumab, adalimumab, bei Zuoluo mab, cetuximab-pekou, cetuximab-bogus, dactylumab, darimumab, ding Tuo mab, ibritumomab, erluzumab, erlotinu3, etamomab, gemtuzumab-ozimab, ji Tuo mab, nesuximab, oxybutylimumab, famomab, trastuzumab, rituximab, toxib, and ibritumomab.
According to some embodiments of the invention, the antibody is selected from the group consisting of rituximab, cetuximab, and alemtuzumab.
According to some embodiments of the invention, the method comprises: after the activation, adoptively transferring the plurality of immune cells to a subject in need thereof.
According to some embodiments of the invention, the subject has a disease associated with the plurality of cells that exhibit the CD 47.
According to some embodiments of the invention, the sirpa-41 BBL fusion protein comprises the sirpa-41 BBL fusion protein of the invention; the polynucleotide or the nucleic acid construct comprises the polynucleotide or the nucleic acid construct of the invention; and said host cell comprises said host cell of the invention.
According to some embodiments of the invention, the plurality of immune cells comprises T cells.
According to some embodiments of the invention, the plurality of immune cells comprises phagocytes.
Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, various exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting as to the necessity.
Drawings
Some embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. Referring now in specific detail to the drawings, it is emphasized that the various details shown are by way of example and for purposes of illustrative discussion of various embodiments of the invention. In this regard, the description taken in conjunction with the several drawings makes apparent to those skilled in the art how the several embodiments of the present invention may be embodied.
In the drawings:
FIG. 1 is a photograph of a western blot analysis of His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) under reduced or non-reduced conditions. After affinity purification, the various proteins, either denatured or non-denatured (250 nanograms per well), were separated on SDS-PAGE electrophoresis gel, as indicated, followed by immunoblotting with an anti-His tag antibody;
FIGS. 2A-2B are multiple photographs of Western blot analysis of the His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) under reduced or non-reduced conditions. After affinity purification, the various proteins (250 nanograms per well) were separated on SDS-PAGE electrophoresis gel with denaturation (fig. 2A) or non-denaturation (fig. 2B), followed by immunoblotting with an antibody against 41 BBL;
FIG. 2C is a photograph of Coomassie blue staining of SDS-PAGE electrophoretic analysis of the His tagged SIRPal alpha-41 BBL (SEQ ID NO: 5) with or without a reduction with deglycosylating enzyme. The His-tagged SIRPalpha-41 BBL band is marked with a small black arrow;
FIGS. 3A to 3B are diagrams for explaining the registration of trade names by BrittsTo determine a plurality of patterns of interaction of the His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) with its corresponding ligand. FIG. 3A illustrates binding to CD 47-A biosensor was preloaded with CD47: fc followed by incubation with either the His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) or PD1-CD70 (SEQ ID NO:6, as a negative control group). FIG. 3B illustrates binding to 41 BB-a biosensor preloaded with 41BB: fc followed by incubation with the His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) or PD1-CD70 (SEQ ID NO:6, as a negative control group);
FIGS. 4A-4B are graphs illustrating the phenotype of multiple indicator receptors on Chinese hamster ovary-K1-wild type cells (CHO-K1-WT) (FIG. 4A) and CHO-KI-CD40 cells (FIG. 4B). Determining the surface manifestation of 41BB and CD47 by immunostaining each cell line with the corresponding antibody, followed by flow cytometry analysis;
FIGS. 5A-5B illustrate binding of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) to CHO-K1-47 cells (FIG. 5A), but not to CHO-K1-WT cells (FIG. 5B). The plurality of cells were incubated with different concentrations of the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) for 30 minutes, followed by immunostaining with a primary antibody to 41BBL and flow cytometry analysis. Values of Geometric Mean Fluorescence Intensity (GMFI) were used to generate a binding graph by GraphPad Prism software; FIG. 6 is a diagram illustrating the promotion of Tumor Necrosis Factor Receptor (TNFR) signaling by the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) demonstrated by interleukin-8 (IL-8) secreted from HT1080-41BB cells in a medium containing Fetal Bovine Serum (FBS);
FIG. 7 is a diagram illustrating the promotion of TNFR signaling by the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) demonstrated by IL-8 secreted from HT1080-41BB cells in serum-free medium;
FIGS. 8A-8E illustrate that His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) triggers 41BB co-stimulatory signaling and enhances T cell activation. FIG. 8A is a graph illustrating the concentration of IL-8 in the supernatant of HT1080-41BB cell culture and the co-culture of HT1080-41BB with CHO cells after treatment with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 8B is a graph illustrating the concentration of IL-8 in the supernatant of HT1080-41BB cell culture and the co-culture of HT1080-41BB with CHO-CD47 cells after treatment with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 8C is a graph illustrating the assessment of T cell activation by CD25 expression, the T cells were cultured in CD47: fc coated 96-well plates for 3 days and treated with anti-CD 3/anti-CD 38 beads at a single optimal concentration, and increased concentration of His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5). FIG. 8D-8E show multiple representative plots (FIG. 8D) and a summary plot (mean.+ -. Standard deviation), two independent donors were taken in two independent experiments, each donor was analyzed in a triplicate fashion FIG. 8E is a multiple plot to illustrate the assessment of T cell activation by the expression of CD25, the T cells isolated from peripheral blood of healthy volunteers were mixed with human colorectal adenocarcinoma epithelial cells (DLD-1), co-cultured, and treated for 3 days with or without His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) at one optimal concentration of anti-CD 3/anti-CD 28 beads;
FIGS. 9A-9C illustrate that SIRPalpha-41 BBL does not have a direct killing effect on MV4-11 cancer cells. FIG. 9A is a graph showing the expression of CD47 on the surface of MV4-11 cells; FIG. 9B illustrates binding of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) to MV4-11 cells. The cells were incubated with Fc-blocker for 15 min on ice, followed by incubation with different concentrations of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) for 30 min, and immunostaining and flow cytometry analysis with anti-41 BBL antibodies. FIG. 9C is a graph showing that MV4-11 cells incubated with different concentrations of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) for up to 72 hours did not show any direct killing effect by Propidium Iodide (PI) staining;
FIGS. 10A-10B illustrate that the His-tagged SIRPalpha-41 BBL promotes secretion of interferon-gamma (INF-gamma) from anti-CD 3 primed human peripheral mononuclear blood cells (PBMCs). FIG. 10A is a graph illustrating the concentration of IFN-gamma detected in culture supernatants of human PBMCs incubated with different concentrations of the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) for 40 hours in the presence of anti-CD 3 or anti-CD 3 plus IL2, as indicated. FIG. 10B is a schematic illustration of the presence or absence of anti-CD 3; and a pattern of IFN-gamma concentrations detected in culture supernatants of human PBMCs co-cultured with human cancer MV4-11 cells and incubated with different concentrations of His-tagged SIRPal alpha-41 BBL protein (SEQ ID NO: 5) for 40 hours, with or without IL2, as indicated;
FIGS. 11A-11L illustrate that SIRPalpha-41 BBL enhances pellet-mediated phagocytosis. FIG. 11A shows a representative gating (gating) strategy for phagocytosis analysis of flow cells. FIG. 11B is a diagram illustrating phagocytosis of B cell lymphoma cell line BJAB by pellet spheres after treatment with a plurality of specified concentrations of His-tagged SIRPal alpha-41 BBL protein (SEQ ID NO: 5). FIG. 11C is a diagram illustrating phagocytosis of a designated B cell lymphoma cell line by pellet obtained from 2 to 3 independent donors with or without incubation with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 11D is a diagram illustrating phagocytosis of a designated B cell lymphoma cell line by pellet after treatment with rituximab (rituximab) with or without His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 11E is a diagram illustrating phagocytosis of a designated cancer cell line by pellet spheres obtained from 3 to 6 independent donors, with or without incubation with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 11F is a diagram illustrating phagocytosis of a designated cancer cell line by pellet spheres after treatment with cetuximab (cetuximab), with or without His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 11G is a diagram illustrating phagocytosis of a designated myeloid leukemia cell line by pellet obtained from 3 independent donors with or without incubation with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) for 2 hours. FIG. 11H is a diagram illustrating phagocytosis of a designated myeloid leukemia cell line by pellet obtained from 3 independent donors with or without incubation with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) for 24 hours. FIG. 11I is a diagram illustrating phagocytosis of human acute promyelocytic leukemia cells (HL 60) and acute myelogenous leukemia cells (MOLM 13) by pellet balls after treatment with a plurality of specified concentrations of His-tagged SIRP alpha-41 BBL protein (SEQ ID NO: 5). FIG. 11J is a diagram illustrating phagocytosis of primary acute myelogenous leukemia cells by pellet spheres after treatment with a plurality of specified concentrations of His-tagged SIRP alpha-41 BBL protein (SEQ ID NO: 5) for 2 hours or 24 hours. FIG. 11K is a graph illustrating phagocytosis (mean.+ -. Standard deviation) of primary acute myelogenous leukemia cells by allogeneic granules after 2 hours or 24 hours incubation with or without 2.5. Mu.g/ml His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 11L shows phagocytosis of DLD1 cancer cells by polymorphonuclear cells treated with soluble SIRPalpha, soluble 41BBL, a combination of both or His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) for 2 hours;
Fig. 12A-12D illustrate that macrophage-mediated phagocytosis is enhanced. FIG. 12A shows a plurality of representative micrographs of macrophages co-cultured with B cell lymphoma cell U2932 pre-stained with V450 after 2 hours incubation with or without His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) and with or without monoclonal antibody rituximab. The attached macrophages are visible in bright field and have V450 labeled cancer cells that are visible as bright cells. The dark arrows indicate living tumor cells. White arrows indicate tumor cells that have been phagocytosed by macrophages. FIG. 12B is a diagram illustrating phagocytosis mediated by macrophages of U2932 cells after treatment with a plurality of specified concentrations of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 12C is a diagram illustrating phagocytosis mediated by macrophages of U2932 cells after treatment with rituximab, with or without the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5). FIG. 12D is a diagram illustrating phagocytosis mediated by macrophages in primary B-cell malignant chronic lymphocytic leukemia incubated with or without His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) and with or without alemtuzumab (alemtuzumab);
FIGS. 13A-13B illustrate that treatment of CT-26 vaccinated mice with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) reduced tumor volume. Fig. 13A is a schematic illustration of an experimental time axis: day 0 at 1x10 6 Mice were inoculated subcutaneously and injected with Phosphate Buffered Saline (PBS) control group, alpha PD1 or SIRP alpha-41 BBL on days 3, 7 and 10. Fig. 13B is a graph illustrating mean ± standard deviation of tumor volumes in three treatment groups);
FIG. 14A-14B illustrate that His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) is effective in treating mice vaccinated with P338 isogenic leukemia tumors. Fig. 14A is a schematic illustration of an experimental time axis: day 0 at 1x10 6 Mice were vaccinated intraperitoneally, and injections of PBS control group, alpha PD1 or SIRP alpha-41 BBL were made on days 1, 3, 5 and 7. FIG. 14B is a diagram illustrating spleen weight in three treatment groups at the time of sacrifice; and/or
FIGS. 15A-15C illustrate that His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) reduces tumor burden in Bone Marrow (BM) of NSG mice vaccinated with human leukemia tumors. Fig. 15A is a schematic illustration of an experimental time axis: mice were irradiated 24 hours prior to MV4-11 cell inoculation, and after 13 days, mice were inoculated with human peripheral mononuclear cells (PBMCs) and treatment was initiated after 4 hours. Each group of 5 animals was dosed by intraperitoneal injection (on days 13, 15, 17 and 19) with His-tagged sirpa-41 BBL protein (SEQ ID NO: 5) (100 μg/injection) or its soluble buffer (PBS) 4 times every 1 day (EOD). Mice were sacrificed 24 hours after the last injection. Fig. 15B shows the number of leukemia cells in bone marrow as determined by using flow cytometry. Fig. 15C shows spleen weight in milligrams.
Detailed Description
In some embodiments of the invention, the invention relates to a SIRPalpha-41 BBL fusion protein and methods of using the SIRPalpha-41 BBL fusion protein.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the various examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
Dual Signaling Proteins (DSPs), also known in the art as signaling proteins (SCPs), are currently known in the art as bifunctional fusion proteins that link an extracellular portion (extracellular amino-terminus) of a first type of membrane protein to an extracellular portion (extracellular carboxy-terminus) of a second type of membrane protein to form a fusion protein having two active sides.
Unexpectedly, a particular fusion protein was found to be advantageously administered to a plurality of subjects suffering from a cancerous disease, depending on the presence of a plurality of tumors having a plurality of tumor-infiltrating lymphocytes (TILs) in a tumor microenvironment and a plurality of tumors having relatively high expression of CD47 on or in the tumor microenvironment.
As will be described below and in the examples section, the present invention has produced a His-tagged SIRPalpha-41 BBL fusion protein (SEQ ID NO: 5) and shows that the fusion protein (SEQ ID NO: 5) comprises two domains and is produced as at least one trimer (experiments 1A-1B, FIGS. 1 and 2A-2C). Next, the inventors demonstrated that the His-tagged sirpa-41 BBL fusion protein (SEQ ID NO: 5) produced retains a functional binding activity to its cognate receptor CD47 and 41BB (experiments 1C-1D, fig. 3A-3B, fig. 4A-4B, and fig. 5A-5B), and can trigger co-stimulation of 41BB and activation of multiple cells exhibiting 41BB (e.g., T cells, peripheral mononuclear cells (PBMCs)), wherein the presence of CD47 enhances this activity (experiments 2-3 and 3A-3B, fig. 6-7, fig. 8A-8E, fig. 9A-9C, and fig. 10A-10B). Furthermore, the inventors demonstrated that the His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) enhances phagocytic uptake of various malignant cell types, including primary malignant cells, by its SIRPalpha domain, particularly in combination therapies with various therapeutic monoclonal antibodies currently in clinical use (experiment 4, FIGS. 11A-11L and FIGS. 12A-12D). The inventors further demonstrated that His-tagged sirpa-41 BBL fusion protein (SEQ ID NO: 5) was effective for the treatment of a variety of tumors as shown in the in vivo mouse tumor model of colon cancer and leukemia (experiments 5 and 5A-5C, fig. 13A-13B, fig. 14A-14B and fig. 15A-15C).
Thus, the present teachings propose a sirpa-41 BBL fusion protein, a polynucleotide encoding the sirpa-41 BBL fusion protein, and a host cell expressing the sirpa-41 BBL fusion protein; and the sirpa-41 BBL fusion proteins are generally used, for example, in the activation of multiple immune cells (by co-stimulation), and in particular in the treatment of a variety of diseases (e.g., cancer) that may benefit from the activation of multiple immune cells.
Thus, according to a first aspect of the invention there is provided a SIRP alpha-41 BBL fusion protein or a variant or fragment of any of said SIRP alpha-41 BBL fusion proteins, or a SIRP alpha-41 BBL fusion protein having at least about 70% homology to the sequence set forth in SEQ ID NO.4, optionally with a linking chain between the SIRP alpha-41 BBL fusion proteins.
According to another aspect of the invention, there is provided a SIRPalpha-41 BBL fusion protein comprising a single amino acid linking chain between SIRPalpha and 41 BBL.
According to another aspect of the invention there is provided a SIRPalpha-41 BBL fusion protein, said SIRPalpha-41 BBL fusion protein being in the form of at least one homotrimer.
According to various specific embodiments of the present invention,
According to specific embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sirpa-41 BBL fusion protein is in the form of at least one homotrimer, each possibility representing a separate embodiment of the invention.
According to various specific embodiments, the at least homotrimer comprises a homotrimer.
According to various specific embodiments, the at least homotrimer comprises a homotetramer.
According to various specific embodiments, the at least homotrimer comprises a homopentamer.
According to various specific embodiments, the at least homotrimer comprises a homohexamer.
Various methods of determining the three-polymerization are well known in the art and include, but are not limited to, SDS-PAGE electrophoresis, raw gel electrophoresis (NATIVE-PAGE), size exclusion high performance chromatography (SEC-HPLC), two-dimensional gel, gel filtration, size exclusion chromatography and multi-angle laser light scattering (SEC MALLS), ultra high speed centrifugation (AUC) Mass Spectrometry (MS), capillary Gel Electrophoresis (CGE).
According to particular embodiments, the at least one homotrimer has a molecular weight of at least 140 kilodaltons, at least 160 kilodaltons, at least 180 kilodaltons, at least 200 kilodaltons, at least 220 kilodaltons, at least 240 kilodaltons, as determined by SDS-PAGE electrophoresis.
According to various specific embodiments, the at least one homotrimer has a molecular weight of at least 140 kilodaltons as determined by SDS-PAGE electrophoresis.
According to various specific embodiments, the at least one homotrimer has a molecular weight of at least 200 kilodaltons as determined by SDS-PAGE electrophoresis.
The term "SIRPA (signal regulatory protein a, also known as CD172 a)" as used herein refers to a polypeptide or a functional homolog of the SIRPA gene (gene number 140885), e.g., a functional fragment thereof. According to particular embodiments, the term "sirpa" refers to a functional homolog of a sirpa polypeptide. According to various specific embodiments, the sirpa is human sirpa. According to a specific embodiment, the sirpa protein refers to a human protein, for example, as provided in GenBank code np_001035111, np_001035112, np_001317657 or np_542970 below.
As used herein, a "functional sirpa" is capable of binding to its cognate receptor CD47[ also known as an integrin-associated protein (IAP) ].
As used herein, the phrase "functional homolog" or "functional fragment" when related to sirpa refers to a portion of a polypeptide that maintains a full-length activity of sirpa, e.g., binding to CD 47.
According to a specific embodiment, the CD47 protein refers to a human protein, for example, as provided in GenBank code np_001768 or np_942088 below.
Multiple assays for testing binding are well known in the artAnd the plurality of assays include, but are not limited to, flow cytometry, biomolecular interaction analysis (BiaCore), registered trade name britesesIs a biological membrane interferometry, high Performance Liquid Chromatography (HPLC).
According to various embodiments, sirpa binds CD47 with a dissociation constant (Kd) of 0.1 to 100 micromoles per liter (μm), 0.1 to 10 μm, 1 to 10 μm, 0.1 to 5 μm, or 1 to 2 μm, as determined by Surface Plasmon Resonance (SPR) analysis, each possibility representing a separate embodiment of the invention.
According to particular embodiments of the invention, sirpa comprises an extracellular domain of the sirpa or a fragment of the extracellular domain.
According to particular embodiments of the invention, the amino acid sequence of SIRPalpha comprises SEQ ID NO 9.
According to specific embodiments of the invention, the amino acid sequence of SIRPalpha consists of SEQ ID NO 9.
According to particular embodiments of the invention, the amino acid sequence of SIRPalpha comprises SEQ ID NO 10.
According to specific embodiments of the invention, the amino acid sequence of SIRPalpha consists of SEQ ID NO 10.
According to particular embodiments of the invention, the amino acid sequence of SIRPalpha comprises SEQ ID NO. 2.
According to specific embodiments of the invention, the amino acid sequence of SIRPalpha consists of SEQ ID NO. 2.
According to particular embodiments of the invention, the amino acid sequence of SIRPalpha comprises SEQ ID NO. 11.
According to specific embodiments of the invention, the amino acid sequence of SIRPalpha consists of SEQ ID NO. 11.
The term "sirpa" also encompasses a plurality of functional homologs (naturally occurring or synthetically/recombinantly produced) that exhibit a desired activity (i.e., binding to CD 47). For example, such homologs may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% similar or homologous to polypeptide SEQ ID NOs 2 or 9; or at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% similar or homologous to a polynucleotide sequence encoding said polypeptide SEQ ID No. 2 or 9 (as described further below).
The similarity or homology of sequences may be determined by using any alignment algorithm for proteins or nucleic acid sequences, such as Blast, clustalW and MUSCLE.
The homologue may also refer to a heterologous homologue, a deletion, insertion or substitution variant comprising an amino acid substitution, as described further below.
According to various embodiments, the sirpa polypeptide may comprise a plurality of amino acid substitutions of the reserved type, as described further below.
According to various embodiments, the amino acid sequence of sirpa comprises 100 to 500 amino acids, 150 to 450 amino acids, 200 to 400 amino acids, 250 to 400 amino acids, 300 to 400, 320 to 420 amino acids, 340 to 350 amino acids, each possibility representing a separate embodiment of the invention.
According to specific embodiments, the sirpa amino acid sequence is 300 to 400 amino acids in length.
According to specific embodiments, the sirpa amino acid sequence is 340 to 450 amino acids in length.
According to specific embodiments, the sirpa amino acid sequence is 343 amino acids in length.
As used herein, the term "41BBL (also known as CD137L and TNFSF 9)" refers to a polypeptide or a functional homolog of the TNFSF9 gene (gene number 8744), e.g., a functional fragment thereof. According to various embodiments, the term "41BBL" refers to a functional homolog of the 41BBL polypeptide. According to various specific embodiments, the 41BBL is a human 41BBL. According to a specific embodiment, the 41BBL protein refers to a human protein, for example, as provided in GenBank code np_003802 below.
According to various embodiments, the 41BBL comprises an extracellular domain of 41BBL or a functional fragment of the extracellular domain.
According to specific embodiments, the amino acid sequence of 41BBL comprises SEQ ID NO. 12.
According to several specific embodiments, the amino acid sequence of 41BBL consists of SEQ ID NO. 12.
According to various specific embodiments, the amino acid sequence of 41BBL comprises SEQ ID NO. 13.
According to several specific embodiments, the amino acid sequence of 41BBL consists of SEQ ID NO. 13.
According to specific embodiments, the amino acid sequence of 41BBL comprises SEQ ID NO. 3.
According to several specific embodiments, the amino acid sequence of 41BBL consists of SEQ ID NO. 3.
According to various specific embodiments, the amino acid sequence of 41BBL comprises SEQ ID NO. 14.
According to various specific embodiments, the amino acid sequence of 41BBL consists of SEQ ID NO. 14.
The term "41BBL" also encompasses a plurality of functional homologs (naturally occurring or synthetically/recombinantly produced) that exhibit a desired activity (as defined below). For example, such homologs may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% similar or homologous to polypeptide SEQ ID NOs 3, 12; or at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% similar or homologous to a polynucleotide sequence encoding said SEQ ID NO 3, 12 (as described further below).
According to various specific embodiments, the 41BBL polypeptide comprises a plurality of remaining amino acid substitutions, as described further below.
According to various embodiments, the amino acid sequence of 41BBL comprises 100 to 300 amino acids, 150 to 250 amino acids, 100 to 250 amino acids, 150 to 220 amino acids, 180 to 220 amino acids, 190 to 210 amino acids, each possibility representing a separate embodiment of the invention.
According to specific embodiments, the 41BBL amino acid sequence is 190 to 210 amino acids in length.
According to specific embodiments, the 41BBL amino acid sequence is 204 amino acids in length.
As used herein, a "functional 41BBL" is capable of at least one of:
(i) Binds to its cognate receptor 41BB (also known as CD 137);
(ii) Activating the signaling pathway of 41BB in an immune cell expressing said 41 BB; and/or
(iii) Activating a plurality of immune cells expressing the 41 BB.
According to specific embodiments, the functional 41BBL is capable of performing (i), (ii), (iii), (i) + (ii), (i) + (iii), (ii) + (iii).
According to specific embodiments, the functional 41BBL is capable of performing (i) + (ii) + (iii).
As used herein, the phrase "functional homolog" or "functional fragment" when referring to 41BBL refers to a portion of a polypeptide that maintains the activity of a full length of 41BBL, e.g., binding to 41BB, activating the signaling pathway of 41BB, activating a plurality of immune cells that express 41 BB.
According to a specific embodiment, the 41BB protein is a human protein, such as provided in GenBank code NP-001552 below.
A number of assays for testing binding are well known in the art and are further described above.
According to various embodiments, each possibility represents a separate embodiment of the claimed invention, 41BBL binds to 41BB with a dissociation constant of about 0.1-1000 nanomoles per liter (nM), 0.1-100 nM, 1-100 nM, or 55.2nM, as determined by SPR.
As used herein, the term "activate" or "activation" refers to the process of stimulating an immune cell (e.g., T cell, B cell, natural killer cell, phagocyte) that results in proliferation, maturation, cytokine production, phagocytosis, and/or induction of regulatory or effector functions of the cell.
According to various embodiments, the activating comprises performing co-stimulation.
As used herein, the term "co-stimulating" or "co-stimulation" refers to the delivery of a secondary antigen-independent stimulation signal (e.g., 41BB signal) to cause activation of the immune cells.
According to various embodiments, the activating comprises suppressing an inhibitory signal (e.g., CD47 signal) to cause activation of the immune cells.
Methods for determining the transmission of a stimulating signal or an inhibiting signal are well known in the art and are also disclosed in the examples section, as follows, and include, but are not limited to, by using binding assays such as BiaCore, HPLC, or flow cytometry; a plurality of enzyme activity assays, such as kinase activity assays; and the expression of a plurality of molecules involved in a signaling cascade by using, for example, polymerase Chain Reaction (PCR), western blot method, immunoprecipitation method, and immunohistochemistry. Additionally or alternatively, an assay for signal transduction (co-stimulation or inhibition) may be accomplished by assessing immune cell activation or function. A number of methods for assessing activation or function of immune cells are well known in the art and include, but are not limited to, a number of proliferation assays, such as CFSE staining, MTS method, alamarblue (alamarblue), deoxyuridine Bromide (BRDU) method, thymine intercalation method; a number of cytotoxicity assays, such as CFSE staining, chromium release, calcein acetyl methyl ester (Calcin AM) methods; a number of assays for cytokine secretion, such as intracellular cytokine staining, enzyme Linked Immunospot (ELISPOT) and enzyme immunoassay (ELISA); markers expressed by multiple activations using flow cytometry, e.g., CD25, CD69, CD137, CD107a, PD1 and CD62L.
According to various embodiments, the determination of the activity or activation of signaling is accomplished in vitro or in vitro, for example in a Mixed Lymphocyte Reaction (MLR), as described further below.
For the same culture conditions, the activity of the signaling or activation or function of the immune cell is typically expressed as compared to signaling, activation or function in a cell of the same species, but is not contacted with the sirpa-41 BBL fusion protein, a polynucleotide encoding the sirpa-41 BBL fusion protein, or a host cell encoding the sirpa-41 BBL fusion protein; or with a carrier control group, which is also referred to as a control group.
The terms "dual signaling protein" and "fusion protein", "chimeric protein" or "chimeric" are used interchangeably herein and refer to an amino acid sequence having two or more portions that are not found together in a single amino acid in nature.
In one embodiment, the invention is directed to a fusion protein comprising sirpa-41 BBL (hereinafter sirpa-41 BBL fusion protein), or a variant or fragment of any of the fusion proteins, optionally with a linking chain therebetween.
Sirpa-41 BBL is a chimeric protein of a Dual Signaling Protein (DSP) that fuses the extracellular domains of two different human membrane proteins. An N-terminal domain is an extracellular domain of human SIRPa (gene: SIRPA), which is a first type of membrane protein, and a C-terminal domain of the chimera is an extracellular domain of human 41BBL (gene: 41 BBL), which is a second type of membrane protein.
According to specific embodiments, the sirpa-41 BBL fusion protein is soluble (i.e., not immobilized on a synthetic or naturally occurring surface).
According to specific embodiments, the sirpa-41 BBL fusion protein is immobilized on an artificially synthesized or naturally occurring surface.
According to particular embodiments, the sirpa-41 BBL does not comprise a linking chain between sirpa and 41 BBL.
In some embodiments, the sirpa-41 BBL comprises a linking chain, which can be any length.
Thus, according to various specific embodiments, the sirpa-41 BBL comprises a linking chain between sirpa and 41 BBL.
Any linking chain known in the art may be used in various specific embodiments of the present invention.
According to specific embodiments, the linking strand may be derived from a plurality of naturally occurring multidomain proteins, or the linking strand is an experimental linking strand, e.g., as described in Chichili et al, (2013), protein Sci.22 (2): 153-167, chen et al, (2013), adv Drug Deliv Rev.65 (10): 1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the link chain is designed using a plurality of link chain design databases and a plurality of computer programs, for example, as described in Chen et al, (2013), adv Drug Deliv rev.65 (10): 1357-1369 and Crasto et al, (2000), protein eng.13 (5): 309-312, the entire contents of which are incorporated herein by reference.
According to various specific embodiments, the linking chain is an artificially synthesized linking chain, such as polyethylene glycol (PEG).
According to specific embodiments, the linking chain is a crystallizable fragment (Fc) domain or a hinge region of an antibody (e.g., immunoglobulin G (IgG), immunoglobulin a (IgA), immunoglobulin D (IgD), or immunoglobulin E (IgE)) or a fragment thereof.
According to other specific embodiments, the linking strand is not a crystallizable fragment domain or a hinge region of an antibody or fragments thereof.
According to various specific embodiments, the link chain may be functional. For example, but not limited to, the linking chain may function to improve foldability and/or stability, improve performance, improve pharmacokinetics, and/or improve the biological activity of the sirpa-41 BBL fusion protein. In another example, the linking strand may function to target the sirpa-41 BBL fusion protein to a particular cell type or location.
According to various embodiments, the linking chain is a polypeptide.
In some embodiments, the sirpa-41 BBL comprises a linking chain that is 1 to 6 amino acids in length.
According to various embodiments, the linking chain consists essentially of glycine and/or serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or 100% glycine and serine).
According to various embodiments, the linking chain is a single amino acid linking chain.
In some embodiments of the invention, the amino acid that links SIRPalpha and 41BBL is glycine, also referred to herein as SIRPalpha-G-41 BBL fusion proteins.
According to specific embodiments, the amino acid sequence of the SIRPalpha-41 BBL fusion protein comprises SEQ ID NO. 1.
According to specific embodiments, the amino acid sequence of the SIRPalpha-41 BBL fusion protein consists of SEQ ID NO. 1.
In some embodiments, the term "SIRPalpha-G-41 BBL" refers to a protein identified by SEQ ID NO: 1:
amino acid sequence of chimeric protein (sirpa-G-41 BBL):
“EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY”G“ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE”
the extracellular domain of human SIRP alpha protein is indicated below, meaning
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY(SEQ ID NO.2)
The extracellular domain of human 41BBL is indicated as follows, i.e.
ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO.3)
According to specific embodiments, the amino acid sequence of SIRPalpha-G-41 BBL is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence set forth in SEQ ID NO. 1, or to a polynucleotide sequence encoding said SEQ ID NO. 1.
In some embodiments, a sirpa-41 BBL fusion protein is provided that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence set forth in SEQ ID No.4, optionally with a linking chain between the sirpa peptide or extracellular domain thereof (ECD) and the 41BBL peptide or extracellular domain thereof, wherein SEQ ID No.4 is:
“EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY”ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE
In some embodiments, there is provided a sirpa-41 BBL as set forth in SEQ ID No.4, optionally having a linking chain between a sirpa peptide or extracellular domain thereof and a 41BBL peptide or extracellular domain thereof, wherein SEQ ID No.4 is:
“EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY”ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE
in additional embodiments, the SIRPA-G-41BBL fusion protein may be a variant and/or derivative of the amino acid sequence shown in SEQ ID NO. 1. Many such variants are known in the art, for example in Weiskopf et al,2013; young Won, et al,2010 and Rabu, et al,2005, incorporated herein by reference as if fully set forth herein.
According to various specific embodiments, the sirpa-41 BBL fusion protein is capable of at least one of:
(i) Binding CD47 and 41BB;
(ii) Activating the signaling pathway of 41BB in an immune cell (e.g., T cell) that expresses 41BB;
(iii) Activating a plurality of immune cells (e.g., T cells) exhibiting 41BB; and/or
(iv) Phagocytosis of a plurality of pathological cells expressing CD47 by a plurality of phagocytes is enhanced compared to in the absence of the sirpa-41 BBL fusion protein.
According to specific embodiments, the sirpa-41 BBL fusion protein is capable of performing (i), (ii), (iii), (iv), (i) + (ii), (i) + (iii), (i) + (iv), (ii) + (iii), (ii) + (iv), (i) + (ii) + (iii), (i) + (ii) + (iv), (ii) + (iii) + (iv).
According to specific embodiments, the sirpa-41 BBL fusion protein is capable of performing (i) + (ii) + (iii) + (iv).
Various methods for determining binding, activation of 41BB signaling pathway, and activation of multiple immune cells are well known in the art and are further described in the various examples section above and below.
According to specific embodiments, the sirpa-41 BBL fusion protein enhances phagocytosis of a plurality of CD47 expressing pathological cells by a plurality of phagocytes.
Various methods for analyzing phagocytosis are known in the art and are also disclosed in experiment 4 in the various examples section below; the plurality of methods include, for example, killing assays, flow cytometry, and/or microscopic assessment (live cell images, fluorescent microscopy, confocal microscopy, electron microscopy).
According to particular embodiments, the enhancement of phagocytosis is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold as compared to the enhancement of phagocytosis in the absence of the sirpa-41 BBL fusion protein of the invention, the polynucleotide encoding the sirpa-41 BBL fusion protein, or the nucleic acid construct, or the host cell expressing the sirpa-41 BBL fusion protein, as determined, for example, by flow cytometry or microscopic evaluation.
According to other specific embodiments, the increase in survival is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to the presence of the sirpa-41 BBL fusion protein of the invention, the polynucleotide encoding the sirpa-41 BBL fusion protein, or the nucleic acid construct, or the host cell expressing the sirpa-41 BBL fusion protein, as determined by, for example, flow cytometry or microscopic evaluation.
Since compositions of some embodiments of the invention (e.g., a fusion protein, a polynucleotide or nucleic acid encoding the fusion protein, or a host cell that has the fusion protein on its surface) are capable of activating immune cells, they can be used in methods of activating immune cells in vitro, and/or in vivo.
Thus, according to one aspect of the present invention there is provided a method for activating a plurality of immune cells, the method comprising: activating the plurality of immune cells in vitro or in vitro in the presence of a sirpa-41 BBL fusion protein, a polynucleotide encoding the sirpa-41 BBL fusion protein, a nucleic acid construct encoding the sirpa-41 BBL fusion protein, or a host cell expressing the sirpa-41 BBL fusion protein.
According to another aspect of the present invention there is provided a method for activating a plurality of T cells, the method comprising: activating a plurality of T cells in vitro or in vitro in the presence of a sirpa-41 BBL fusion protein and a plurality of cells expressing CD 47.
According to another aspect of the present invention there is provided a method for activating a plurality of phagocytes, the method comprising: activating a plurality of phagocytes in vitro in the presence of a sirpa-41 BBL fusion protein and a plurality of cells expressing CD 47.
According to specific embodiments, the plurality of immune cells express 41BB.
According to various specific embodiments, the plurality of immune cells comprises peripheral mononuclear cells (PBMCs).
As used herein, the term "peripheral mononuclear cells (PBMCs)" refers to a blood cell having a single nucleus and includes lymphocytes, mononucleated spheres, and Dendritic Cells (DCs).
According to various embodiments, the peripheral mononuclear cells are selected from the group consisting of Dendritic Cells (DCs), T cells, B cells, natural Killer T (NKT) cells.
According to various embodiments, the peripheral mononuclear cells comprise T cells, B cells, natural killer cells, and natural killer T cells.
Methods for obtaining peripheral mononuclear cells are well known in the art, such as whole blood from a subject and collecting in a container containing an anticoagulant (e.g., heparin or citrate); and performing blood cell separation. As follows, according to various embodiments, at least one type of peripheral mononuclear cell is purified from peripheral blood. Methods and reagents for purifying peripheral mononuclear cells from whole blood are known to those skilled in the art, such as white blood cell separation, sedimentation, density gradient centrifugation (e.g. ficoll), centrifugal elutriation, fractionation, chemical lysis of e.g. erythrocytes (e.g. by ACK), screening for specific cell types by using labels on cell surfaces (e.g. using Fluorescence Activated Cell Sorting (FACS) or magnetic cell separation techniques, such as obtained from commercial sources such as Invitrogen, stem cell technology (Stemcell Technologies), camptotheca (Cellpro), advanced magnetism (Advanced Magnetics) or spurious (Miltenyi Biotec)), and removal of specific cell types may be achieved by methods such as eradication (e.g. killing) with specific antibodies, or by affinity-based purification based on negative selection (e.g. using magnetic cell separation techniques, fluorescence activated cell sorting and/or capture type labels). Such methods are described, for example, in volumes 1 to 4 of the experimental immunology handbook (d.n.weir, editor) and flow cytometry and cell sorting (a.radburuch, editor, springer Verlag, 2000).
According to specific embodiments, the plurality of immune cells comprises tumor-infiltrating lymphocytes.
As used herein, the term "tumor-infiltrating lymphocytes (TILs)" refers to mononuclear leukocytes that leave the blood stream and migrate to a tumor.
According to various embodiments, the tumor-infiltrating lymphocytes are selected from the group consisting of T cells, B cells, natural killer T cells.
Various methods for obtaining tumor-infiltrating lymphocytes are well known in the art, for example, obtaining a tumor sample from a subject by, for example, biopsy or cadaver testing and preparing a single cell suspension thereof. The single cell suspension may be obtained in any suitable manner, for example mechanically (e.g., by disintegrating the tumor using a decomposer under the trade name gentleMACS (TM), spurious, obutyrn, collif (calif)) or enzymatically (e.g., collagenase or dnase). At least one type of said tumor-infiltrating lymphocytes can be purified from a cell suspension as follows. Methods and reagents for purifying tumor-infiltrating lymphocytes of the desired type are known to those of skill in the art, such as by screening for a variety of specific cell types using a variety of cell surface markers (e.g., by using fluorescent activated cell sorters or magnetic cell separation techniques, such as those obtained from commercial invitro life technologies, stem cell technology, camptotheca, advanced magnetism, or spurious techniques), and removal of a variety of specific cell types can be accomplished by a variety of methods, such as eradication (e.g., killing) with a variety of specific antibodies, or by affinity-based purification based on negative selection (e.g., using magnetic cell separation techniques, fluorescent activated cell sorters, and/or capture-type ELISA markers). Such methods are described, for example, in volumes 1 to 4 of the experimental immunology handbook (d.n.weir, editor) and flow cytometry and cell sorting (a.radburuch, editor, springer Verlag, 2000).
According to a number of specific embodiments, the plurality of immune cells comprises phagocytes.
As used herein, the term "phagocyte" refers to a cell that is capable of phagocytosis and includes both specific and non-specific phagocytes. A number of methods for assaying phagocytosis are well known in the art and are further disclosed in the context. According to various embodiments, the phagocytes are selected from the group consisting of mononuclear spheres, dendritic cells, and granulosa spheres.
According to various specific embodiments, the phagocytes comprise pellet spheres.
According to various specific embodiments, the phagocytes comprise mononuclear spheres.
According to various specific embodiments, the immune cells comprise a mononuclear sphere.
According to various embodiments, the term "mononuclear sphere" refers to both circulating mononuclear spheres and macrophages (also referred to as mononuclear phagocytes) present in a tissue.
According to various specific embodiments, the mononuclear sphere comprises macrophages. Typically, the cell surface phenotype of the macrophage includes CD14, CD40, CD11b, CD64, F4/80 (mouse)/EMR 1 (human), lysozyme M, MAC-1/MAC-3 and CD68.
According to various specific embodiments, the mononuclear sphere comprises a circulating mononuclear sphere. Typically, the phenotype of the cell surface of the circulating mononuclear sphere includes CD14 and CD16 (e.g., CD14++ CD16-, CD14+CD16++, CD14++ CD 16-).
According to various specific embodiments, the immune cells comprise dendritic cells.
As used herein, the term "Dendritic Cells (DCs)" refers to any member of a different group of morphologically similar cell types found in lymphoid or non-lymphoid tissues. The dendritic cells are a class of specific antigen presenting cells and have a strong capacity for sensitizing Human Leukocyte Antigen (HLA) -restricted T cells. For example, the dendritic cells include plasmacytoid dendritic cells, bone marrow dendritic cells (including immature and mature dendritic cells), langerhans' cells, staggered dendritic cells, and vesicular dendritic cells. The dendritic cells can be identified by function, or by phenotype, in particular by phenotype of the cell surface. These cells are characterized by their unique morphology with multiple veil-like projections on the cell surface, the ability to express antigen to moderate to high levels on the surface HLA of the second type, and in particular to present antigen to multiple naive T cells (see Steinman R, et al, ann. Rev. Immunol.1991; 9:271-196). Typically, the phenotype of the cell surface of the dendritic cell comprises CD1a+, CD4+, CD86+ or HLA-DR. The term dendritic cells includes both immature dendritic cells and mature dendritic cells.
According to a number of specific embodiments, the plurality of immune cells comprises a pellet of particles.
As used herein, the term "pellet" refers to polytype leukocytes characterized by the presence of multiple particles in their cytoplasm.
According to various specific embodiments, the pellet comprises neutrophils.
According to various specific embodiments, the pellet comprises mast cells.
According to various specific embodiments, the immune cells comprise T cells.
As used herein, the term "T cell" refers to a differentiated lymphocyte having cd3+, T Cell Receptor (TCR) + with a cd4+ or cd8+ phenotype. The T cell may be an active T cell or a regulatory T cell.
As used herein, the term "active T cell" refers to a T cell that is capable of activating or directing other immune cells, e.g., by making cytokines or has cytotoxic activity, e.g., cd4+, th1/Th2, cd8+ cytotoxic T lymphocytes
As used herein, the term "regulatory T cell" or "Treg" refers to a T cell that negatively regulates the activation of other T cells, including active T cells, as well as cells of the innate immune system. The regulatory T cells are characterized by maintaining suppression of the response to the effector T cells. According to various embodiments, the regulatory T cell is a cd4+cd25+foxp3+ T cell.
According to specific embodiments, the T cells are cd4+ T cells.
According to other specific embodiments, the T cells are cd8+ T cells.
According to various specific embodiments, the T cell is a memory T cell. Non-limiting examples of such memory T cells include active memory T cells having a CD3+/CD4+/CD45RA-/CCR 7-phenotype, central memory T cells having a CD3+/CD4+/CD45RA-/CCR7+ phenotype, active memory CD8+ T cells having a CD3+/CD8+ CD45RA-/CCR 7-phenotype, and central memory CD8+ T cells having a CD3+/CD8+ CD45RA-/CCR7+ phenotype.
According to particular embodiments, the T cell comprises an engineered T cell transduced with a nucleic acid sequence encoding a performance product of interest.
According to various embodiments, the expression product of interest is a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR).
As used herein, the phrase "transduced with a nucleic acid sequence encoding a T cell receptor" or "transduced with a nucleic acid sequence encoding a T cell receptor" refers to cloning of variant alpha and beta chains of T cells with specificity for a desired antigen presented in the context of the Major Histocompatibility Complex (MHC). Several methods of transduction with such T cell receptors are well known in the art and are described, for example, in Nicholson et al adv hemalo 2012;2012:404081; wang and Rivi re Cancer Gene Ther.2015Mar;22 (2) 85-94); and polymers et al Cancer Gene Therapy (2002) 9,613-623.
As used herein, the phrase "transduced with a nucleic acid sequence encoding a chimeric antigen receptor" or "transduced with a nucleic acid sequence encoding a chimeric antigen receptor" refers to cloning a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR), wherein the chimeric antigen receptor comprises an antigen recognition portion and a T cell activation portion. The Chimeric Antigen Receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen binding domain (e.g., a single chain variable fragment (scFv)) of an antibody associated with a T cell signaling or T cell activation domain. Several methods of transduction with the chimeric antigen receptor are well known in the art and are described, for example, in Davila et al oncoimmunology 2012dec 1;1 (9) 1577-1583; wang and Rivi re Cancer Gene Ther.2015Mar;22 (2) 85-94); maus et al blood.2014apr 24;123 (17) 2625-35; porter DL The New England journal of media.2011, 365 (8): 725-733; jackson HJ, nat Rev Clin oncol.2016;13 (6) 370-383; globerson-Levin et al mol ter 2014;22 (5) 1029-1038.
According to a number of specific embodiments, the plurality of immune cells comprises B cells.
As used herein, the term "B cell" refers to a lymphocyte having a B Cell Receptor (BCR) +, cd19+ and/or b220+ phenotype. The B cells are characterized by their ability to bind to a specific antigen and elicit a humoral immune response.
According to various specific embodiments, the plurality of immune cells comprises natural killer cells.
As used herein, the term "natural killer cell" refers to a plurality of differentiated lymphocytes having a cd16+cd56+ and/or cd57+ TCR-phenotype. The natural killer cells are characterized by an ability to bind and kill a plurality of cells incapable of self-expression of MHC/HLA antigens by activation of a plurality of specific cytolytic enzymes, an ability to kill a plurality of tumor cells or other diseased cells that exhibit a ligand for a natural killer activation receptor, and an ability to release a plurality of protein molecules called cytokines that stimulate or inhibit an immune response.
According to specific embodiments, the plurality of immune cells comprises natural killer T cells.
As used herein, the term "natural killer T cell" refers to a specific group of T cells that exhibit half of the mutated αβ T cell receptor and exhibit various molecular markers typically associated with the natural killer cells, e.g., NK1.1. The natural killer T cells include NK1.1+ and NK 1.1-and CD4+, CD4-, CD8+ and CD 8-cells. The T cell receptor on the natural cell is unique in that it recognizes a plurality of carbohydrate antigens presented by MHC-like molecule CD1d of the first type. The natural killer T cells may have a protective or deleterious effect due to their ability to produce a variety of cytokines for promoting inflammatory reactions or immune tolerance.
According to specific embodiments, the plurality of immune cells is obtained from a healthy subject.
According to specific embodiments, the plurality of immune cells is obtained from a subject suffering from a disorder (e.g., cancer).
According to specific embodiments, the activation is performed in the presence of a plurality of cells exhibiting CD47 or exogenous CD 47.
According to specific embodiments, the activation is performed in the presence of exogenous CD 47.
According to various specific embodiments, the exogenous CD47 is soluble.
According to other embodiments, the exogenous CD47 is immobilized on a solid support.
According to specific embodiments, the activation is performed in the presence of a plurality of cells exhibiting CD 47.
According to specific embodiments, the plurality of cells exhibiting CD47 comprises diseased (diseased) cells.
According to a number of specific embodiments, the plurality of CD47 expressing cells comprises cancer cells.
According to various embodiments, the activation is in the presence of a stimulus capable of delivering at least a primary activation signal [ e.g., the binding of the T Cell Receptor (TCR) to the Major Histocompatibility Complex (MHC)/peptide complex) on the Antigen Presenting Cell (APC) ] resulting in proliferation, maturation of cells, manufacture of cytokines, phagocytosis and/or induction of regulatory or functional functions of immune cells. According to various embodiments, the stimulus may also deliver a secondary co-stimulatory signal.
Methods for determining a dose of the stimulating agent and a ratio between the stimulating agent and the plurality of immune cells are well within the ability of those skilled in the art and therefore are not described herein.
The stimulatory agent may activate the plurality of immune cells in an antigen-dependent or an antigen-independent (i.e., polyclonal) manner.
According to various embodiments, the stimulating agent comprises an antigen-non-specific stimulus.
Such non-specific stimuli are known to those skilled in the art. Thus, as a non-limiting example, when the plurality of immune cells comprises T cells, the antigen-non-specific stimulus can act as an agent capable of binding to a T cell surface structure, as well as induce polyclonal stimulation of the T cells, such as, but not limited to, an anti-CD 3 antibody that binds to a co-stimulatory protein, such as an anti-CD 28 antibody. Other non-limiting examples include anti-CD 2, anti-CD 137, anti-CD 134, ligands for multiple Notch such as Delta-like 1/4, jagged 1/2, either alone or in multiple combinations with the anti-CD 3. Other agents that may induce polyclonal stimulation of T cells include, but are not limited to, mitogens, phytohemagglutinin (PHA), phorbol ester-Ionomycin (PMA-Ionomycin), CEB, sitostatin (CytoStim) (spurious tenitin). According to various embodiments, the antigen-non-specific stimulus comprises anti-CD 3 and anti-CD 28 antibodies. According to specific embodiments, the T cell stimulators comprise anti-CD 3 and anti-CD 28 coated beads, such as CD3CD28 macsibads obtained from spurious technology.
According to various embodiments, the stimulating agent comprises an antigen-specific stimulus.
Non-limiting examples of antigen-specific T cell stimulators include an antigen-loaded antigen presenting cell [ APC, e.g., dendritic cell ] and a peptide-loaded recombinant MHC. Thus, for example, a T cell stimulator may be a dendritic cell preloaded with a desired antigen or transfected with mRNA encoding the desired antigen.
According to various embodiments, the antigen is a cancer antigen.
As used herein, the term "cancer antigen" refers to an antigen that is over-represented by a cancer cell or is represented only by the cancer cell relative to a non-cancer cell. A cancer antigen may be a known cancer antigen or a new specific antigen (i.e., neoantigen) produced in a cancer cell.
A number of non-limiting examples for known multiple cancer antigens include: MAGE-AI, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A7, MAGE-AS, MAGE-A9, MAGE-AIO, MAGE-All, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-l, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-Cl/CT7, MAGE-C2, NY-ES0-1, LAGE-1, SSX-2 (HOM-MEL-40), MAGE-C2, NY-ES-1, SSX-2 (HOM-MEL-40) SSX-3, SSX-4, SSX-5, SCP-1 and XAGE, melanocyte differentiation antigen, p53, ras, CEA, MUCI, PMSA, PSA, tyrosinase, melan-A, MART-I, gplOO, gp75, alphA-Actin-4 (alphaactinin-4), bcr-Abl fusion protein, casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-l, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucose transferase AS fusion protein, HLa-A2, HLa-All, hsp70-2, KIAA0205, mart2, mum-2, mum-3, neo-PAP, tropomyosin-I, OS-9, pml-RAR alpha fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, gnTV, herv-K-mel, NA-88, SP17, and TRP2-Int2, (MART-I), E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human herpes virus antigen, EBNA, human Papilloma Virus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, plSOerbB-3, C-met, nm-23Hl, PSA, TAG-72-4, CA19-9, CA 72-4, CAM 17.1, nuMa, K-ras, alpha fetal protein, 13HCG, BCA225, BTA, CA125, CA 15-3 (CA 27.29\BCA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, C0-029, C-5, 0250, ga733 (Ag 175), HTM-50, MG7, MG 6, 35A-35, TAP 6, TAG 2, or TAG-35 protein binding thereto.
Other tumor antigens that are displayed are well known in the art (see, e.g., W000/20581;Cancer Vaccines and Immunotherapy (2000) Eds Stern, beverley and Carroll, cambridge University Press, cambridge). The sequences of these tumor antigens are not only readily available from a number of public databases, but are also found in International patent application publication Nos. WO 1992/020356 AI, WO 1994/005304 AI, WO 1994/023431 AI, WO 1995/020974 AI, WO 1995/023874 AI and WO 1996/026214 AI.
Alternatively or additionally, a plurality of cancer cells obtained from a subject by, for example, biopsy is used for the identification of a tumor antigen.
Thus, according to various embodiments, the stimulating agent comprises a cancer cell.
According to particular embodiments, the activation is performed in the presence of an anticancer agent.
According to specific embodiments, the purification of the plurality of tumor cells is performed after the activation.
Thus, the present invention also contemplates the isolation of a plurality of immune cells obtained according to the methods of the present invention.
According to particular embodiments, the plurality of immune cells used and/or obtained according to the present invention may be freshly isolated, stored, e.g., frozen (i.e., frozen), for future use at liquid nitrogen temperatures at any stage over a long period of time (e.g., month, year).
A number of methods of performing such cryopreservation are well known to those of ordinary skill in the art and are disclosed, for example, in international patent application nos. WO2007054160 and WO2001039594 and U.S. patent publication No. US 20120149108.
According to various embodiments, a plurality of cells obtained according to the present invention may be stored in a cell bank or a repository or a storage facility.
Thus, the present teachings further contemplate the use of the isolated immune cells and methods of the present invention, as, but not limited to, a source for adoptive immune cell therapy for a variety of diseases that may benefit from activating immune cells, such as hyperproliferative diseases; and a disease associated with immunosuppression and infection.
Thus, according to various embodiments, a method of the present invention comprises: adoptive transfer of the plurality of immune cells to a subject in need thereof following the activating.
According to specific embodiments, immune cells obtained according to methods of the invention are provided for use in an adoptive cell therapy.
The plurality of cells used in accordance with the various embodiments of the present invention may be autologous or non-autologous; they may be isogenic or non-isogenic: is allogeneic or xenogeneic to the subject; each possibility represents a separate embodiment of the invention.
The present teachings also contemplate the use of compositions of the invention (e.g., a fusion protein, a polynucleotide or nucleic acid construct encoding the fusion protein, or a host cell expressing the fusion protein) in methods of treating a disease that may benefit from activating immune cells.
Thus, according to another aspect of the present invention there is provided a method of treating a disease that may benefit from activating a plurality of immune cells, the method comprising: administering the sirpa-41 BBL fusion protein, a polynucleotide or nucleic acid construct encoding the sirpa-41 BBL fusion protein, or a host cell expressing the sirpa-41 BBL fusion protein to a subject in need thereof.
According to another aspect of the invention there is provided the SIRPalpha-41 BBL fusion protein, a polynucleotide or nucleic acid construct encoding the SIRPalpha-41 BBL fusion protein or a host cell expressing the SIRPalpha-41 BBL fusion protein for use in the treatment of a disease that may benefit from activating a plurality of immune cells.
The term "treating" or "treatment" refers to inhibiting, preventing, arresting the development of a disorder (disease, disorder, or body condition) and/or causing a reduction, alleviation or regression of a disorder or symptoms of a disorder. Those skilled in the art will appreciate that various methods and assays can be used to assess the progression of a disorder, and similarly, various methods and assays can be used to assess the reduction, alleviation or regression of a disorder.
As used herein, the term "subject" includes mammals, such as humans, at any age and of any sex. According to particular embodiments, the term "subject" refers to a subject having a disorder (disease, disorder, or body condition). According to specific embodiments, the term encompasses individuals at risk of developing a disorder.
According to specific embodiments, the subject suffers from a disease associated with the plurality of cells that exhibit the CD47.
According to specific embodiments, the plurality of diseased cells of the subject exhibit CD47.
As used herein, the phrase "a disease that can benefit from activating multiple immune cells" refers to a disease in which the immune response of a subject may be sufficient to at least ameliorate symptoms of the disease or delay the onset of symptoms, however, for any reason, the immune response of the subject is not optimal.
A number of non-limiting examples of diseases that may benefit from activation of multiple immune cells include hyperproliferative diseases, multiple diseases associated with immunosuppression, drugs (e.g., mTOR inhibitors, calcineurin inhibitors, steroids) and immunosuppression caused by infection.
According to various embodiments, the disease comprises a hyperproliferative disease.
According to specific embodiments, the hyperproliferative disease comprises sclerosing or fibrosis, idiopathic pulmonary fibrosis, psoriasis, systemic sclerosis/scleroderma, primary cholangitis, primary sclerosing cholangitis, liver fibrosis, prevention of radiation-induced pulmonary fibrosis, myelofibrosis or retroperitoneal fibrosis.
According to various specific embodiments, the hyperproliferative disease comprises cancer.
Thus, according to another aspect of the present invention there is provided a method for treating cancer, the method comprising: administering the sirpa-41 BBL fusion protein to a subject in need thereof.
As used herein, the term cancer includes both malignant and premalignant cancers.
With respect to the premalignant or benign forms of cancer, alternatively, compositions and methods thereof may be applied to prevent the progression of premalignant cancer to a malignant form.
The plurality of cancers that may be treated by the plurality of methods of some embodiments of the invention may be any solid or non-solid cancer and/or metastatic cancer.
According to various specific embodiments, the cancer comprises a malignant cancer.
Examples of such cancers may include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include: squamous cell carcinoma, lung cancer (including small-cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer (kidney or renal cancer), liver cancer (liver cancer), prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatic carcinoma), and various types of head and neck cancer, and B-cell lymphomas (including mild/follicular non-hodgkin's lymphoma (NHL), burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), small Lymphocytic (SL) NHL, moderate/follicular NHL, moderate diffuse NHL, severe immunoblastic NHL, NHL-cleavage, NHL-related lymphomas, and severe lymphoblastic lymphoma; t cell lymphoma, hodgkin's lymphoma; chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); acute Myelogenous Leukemia (AML), acute Promyelocytic Leukemia (APL), hairy cell leukemia; chronic Myelogenous Leukemia (CML); and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with mole-type hamartoma, oedema (e.g., associated with brain tumors), and miglites syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. A plurality of cancerous conditions amenable to treatment of the present invention include metastatic cancer.
According to various specific embodiments, the cancer comprises a pre-cancerous cancer.
A variety of precancerous cancers (or precancers) are well known and well characterized in the art (e.g., see Berman JJ. And Henson DE.,2003. Classification of a variety of precancers: a metadata method. BMC Med Inform Decis Mak.3: 8). The various classifications of the various pre-cancerous cancers amenable to treatment by the methods of the present invention include: obtaining small or microscopic pre-cancerous cancers, obtaining large lesions with nuclear allotypes, obtaining pre-lesions with genetic proliferative syndromes that progress to cancer, and obtaining diffuse hyperplasia and diffuse metaplasia (metaplasia). Examples of such small or microscopic pre-cancerous cancers include: cervical severe squamous intraepithelial lesions (HGSIL), anal Intraepithelial Neoplasia (AIN), vocal cord dysplasia, abnormal crypt (colon), prostatic Intraepithelial Neoplasia (PIN). Examples of the obtaining of large lesions with nuclear heterogeneity include: tubular adenomas, angioimmunoblastic lymphoadenoses are associated with abnormal proteinemia (AILD), atypical meningiomas, stomach meats, large plaque parapsoriasis, myelodysplasias, transitional cell carcinoma in situ, persistent anaemia is associated with excessive numbers of blastomas, and schneider's papilloma. Examples of such pre-lesions that accompany the progression to the genetic proliferative syndrome of cancer include: atypical nevus syndrome, C-cell adenomatosis, and Multiple Endocrine Adenomatosis (MEA). Examples of the obtaining diffuse hyperplasia include: AIDS, atypical lymphoproliferation, paget's disease of bone, post-transplant lymphoproliferative disorder, and ulcerative colitis.
In some embodiments of the invention, the plurality of diseases treated by a fusion protein comprising SIRPalpha or an extracellular domain thereof and 41BBL or an extracellular domain thereof, such as SIRPalpha-G-41 BBL, are: leukemia, chronic myelomonocytic leukemia (CMML), chronic Myelogenous Leukemia (CML), acute Myelogenous Leukemia (AML), non-hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), B-cell chronic lymphocytic leukemia (B-CLL), mantle Cell Lymphoma (MCL), follicular Lymphoma (FL), marginal Zone Lymphoma (MZL), precursor-B cell acute lymphoblastic leukemia (pre-B ALL), leiomyosarcoma, ovarian cancer, breast cancer, colon cancer, bladder cancer, glioblastoma, hepatocellular carcinoma, prostate cancer, acute Myelogenous Leukemia (AML), multiple myeloma, non-small cell lung cancer (NSCLC), colorectal cancer, melanoma, head and neck cancer, marginal zone B-cell lymphoma, pancreatic ductal adenocarcinoma, brain cancer.
According to some embodiments of the invention, the plurality of specified diseases to be treated by a fusion protein comprising sirpa or an extracellular domain thereof and 41BBL or an extracellular domain thereof, such as sirpa-G-41 BBL, are: acute myelogenous leukemia, bladder cancer, breast cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, diffuse large B-cell lymphoma, epithelial ovarian cancer, epithelial tumors, fallopian tube cancer, follicular lymphoma, glioblastoma multiforme, hepatocellular carcinoma, head and neck cancer, leukemia, lymphoma, mantle cell lymphoma, melanoma, mesothelioma, multiple myeloma, nasopharyngeal carcinoma, non-hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, prostate cancer, renal cell carcinoma.
According to various embodiments, the cancer is selected from the group consisting of lymphoma, leukemia, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, and squamous cell carcinoma.
According to various embodiments, the cancer is selected from the group consisting of lymphoma, carcinoma, and leukemia.
According to various specific embodiments, the cancer is colon cancer.
According to various specific embodiments, the cancer is ovarian cancer.
According to specific embodiments, the cancer is lung cancer.
According to specific embodiments, the cancer is a head and neck cancer.
According to specific embodiments, the cancer is leukemia.
According to a number of specific embodiments of the present invention, the leukemia is selected from the group consisting of acute non-lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute promyelocytic leukemia, adult T-cell leukemia, leukopenia leukemia, basophilic leukemia, embryogenic leukemia, bovine leukemia, chronic myelogenous leukemia, skin leukemia, embryogenic leukemia, eosinophilic leukemia, grosven leukemia, hairy cell leukemia, hemangioblastic leukemia, hematoblast leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenia, leukemia lymphoblastic leukemia (lymphatic leukemia), lymphoblastic leukemia, lymphosarcoma cell leukemia (lymphoid leukemia), lymphosarcoma cell leukemia, mast cell leukemia, megakaryoblastic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelogenous leukemia, myelomonocytic leukemia, internal grignard leukemia, plasma cell leukemia (plasma cell leukemia), plasma cell leukemia (plasmacytic leukemia), pre-myelogenous leukemia, li-delta cell leukemia, schlemn leukemia, stem cell leukemia, sub-leucomatoid leukemia, and undifferentiated cell leukemia.
According to specific embodiments, the leukemia is promyelocytic leukemia, acute myelogenous leukemia, or chronic myelogenous leukemia.
According to various specific embodiments, the cancer is lymphoma.
According to specific embodiments, the lymphoma is a B-cell lymphoma.
According to specific embodiments, the lymphoma is T-cell lymphoma.
According to other specific embodiments, the lymphoma is hodgkin's lymphoma.
According to specific embodiments, the lymphoma is non-hodgkin's lymphoma.
According to specific embodiments, the non-hodgkin's lymphoma is selected from the group consisting of aggressive NHL, altered NHL, low-malignant NHL, recurrent NHL, refractory NHL, mild non-hodgkin's lymphoma, follicular lymphoma, large cell lymphoma, B-cell lymphoma, T-cell lymphoma, mantle cell lymphoma, bozier's lymphoma, natural killer cell lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic and cutaneous T-cell carcinoma, including mycosis fungoides/sezary syndrome.
According to specific embodiments, the cancer is multiple myeloma.
According to specific embodiments, the multiple myeloma is selected from multiple myeloma cancers from which kappa-type light chains and/or lambda-type light chains are made; aggressive multiple myeloma, including primary Plasma Cell Leukemia (PCL); benign plasma cell diseases such as single-strain gammaglobulinemia (MGUS), giant globulinemia (WM, also known as lymphoplasmacytoid lymphoma) that may progress to multiple myeloma; smoldering Multiple Myeloma (SMM), painless multiple bone following tumors, and also multiple pre-malignant forms of myeloma that may develop into multiple myeloma; primary starch deposition.
Suggested SIRPalpha-41 BBL mode of action
In one embodiment of the invention, the chimeric sirpa-41 BBL can be used to treat cancer by the following possible modes of action:
due to the relatively high expression of CD47 on the surface of multiple tumor cells and in a tumor microenvironment, the sirpa portion of the sirpa-41 BBL chimera targets the molecule to tumors and multiple metastasis sites and binds the chimera to CD47 within the tumor microenvironment;
targeting the chimera to the plurality of tumor cells or/and the tumor microenvironment will promote an increase in sirpa-41 BBL concentration in the tumor microenvironment, and subsequent oligomerization of the 4-1BBL portion of the chimera at the tumor site. Because the oligomerization of 4-1BBL is an essential step for the signaling of 4-1BB, this binding and oligomerization of 4-1BBL will deliver a 4-1BB costimulatory signal that promotes activation of T cells, B cells, natural killer cells, particularly Tumor Infiltrating Lymphocytes (TILs) and other immune cells at the tumor site in order to kill multiple cancer cells;
In addition to the 41BBL-41BB co-stimulatory signal, binding of the sirpa moiety of the chimera in the tumor site to CD47 will compete with endogenous sirpa expressed on multiple macrophages and dendritic cells, thus eliminating inhibition of these cells and further causing multiple tumor cell phagocytosis and activation of multiple dendritic cells and T cells in the tumor microenvironment.
The effect of the multiple SIRPA-41BBL is expected to produce a synergistic effect on the activation of TILs, dendritic cells and macrophages in the tumor microenvironment, which is expected to be more specific and potent than the effect of each peptide or its extracellular domain alone and when two different peptides or their extracellular domains are used in combination.
Thus, according to specific embodiments, the cancer is defined as being in the presence of multiple tumors with tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment and/or multiple tumors exhibiting CD47 in the tumor microenvironment.
According to specific embodiments, the cancer is defined as being in the presence of a plurality of tumors having tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment and/or a plurality of tumors having a relatively high manifestation of CD47 in the tumor microenvironment.
According to specific embodiments, the plurality of cells of the cancer exhibit CD47.
According to specific embodiments, the disease comprises a disease associated with immunosuppression or immunosuppression by drugs (e.g., mTOR inhibitors, calcineurin inhibitors, steroids).
According to specific embodiments, the disease comprises Human Immunodeficiency Virus (HIV), measles, influenza, lymphocytic choriomeningitis (LCCM), respiratory fusion virus (RSV), human rhinovirus, human herpesvirus (EBV), cytomegalovirus (CMV), a picovirus.
According to various embodiments, the disease comprises an infection.
As used herein, the term "infection" of an "infectious disease" refers to a disease induced by a pathogen. Specific examples of such pathogens include: viral pathogens, bacterial pathogens, such as intracellular mycobacterial pathogens (e.g., mycobacterium tuberculosis), intracellular bacterial pathogens (e.g., listeria monocytogenes), or intracellular protozoan pathogens (e.g., leishmania and trypanosoma).
A number of specific types of such viral pathogens causing infectious diseases that may be treated in accordance with the teachings of the present invention include, but are not limited to, retrovirus, circovirus, provirus, papovavirus, adenovirus, herpes virus, iridovirus, poxvirus, hepadnavirus, picornavirus, calicivirus, togavirus, flaviviridae, reovirus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, coronavirus, arenavirus, and filovirus.
A number of specific examples of viral infections that may be treated in accordance with the teachings of the present invention include, but are not limited to, human Immunodeficiency Virus (HIV) -induced acquired immunodeficiency syndrome (AIDS), influenza, rhinovirus infections, viral meningitis, human herpes virus (EBV) infections, hepatitis a, B or C virus infections, measles, mastovirus infections/warts, cytomegalovirus (CMV), herpes simplex virus infections, yellow fever, ebola virus infections, rabies, and the like.
According to particular embodiments, compositions of the invention (e.g., sirpa-41 BBL fusion proteins, polynucleotides or nucleic acid constructs encoding the sirpa-41 BBL fusion proteins, and/or host cells expressing the sirpa-41 BBL fusion proteins) may be combined with other established or experimental treatment protocols including, but not limited to, analgesics, chemotherapeutics, radiotherapeutic agents, cytotoxic therapies (conditioning), hormonal therapies, antibodies, and other treatment protocols known in the art (e.g., surgery) for administration to a subject to treat a disease (e.g., cancer) that may benefit from activation of multiple immune cells.
According to particular embodiments, compositions of the invention (e.g., sirpa-41 BBL fusion proteins, polynucleotides or nucleic acid constructs encoding the sirpa-41 BBL fusion proteins, and/or host cells expressing the sirpa-41 BBL fusion proteins) can be combined with adoptive cell transplantation, such as, but not limited to, transplantation of bone marrow cells, hematopoietic stem cells, peripheral mononuclear cells (PBMCs), umbilical cord blood stem cells, and/or induced multifunctional stem cells, for administration to a subject.
According to various embodiments, the therapeutic agent combined with and administered to the compositions of the present invention comprises an anti-cancer agent.
Thus, according to another aspect of the present invention there is provided a method of treating cancer, the method comprising: an anticancer agent; and a SIRPalpha-41 BBL fusion protein, a polynucleotide encoding the SIRPalpha-41 BBL fusion protein, a nucleic acid construct encoding the SIRPalpha-41 BBL fusion protein, and/or a host cell expressing the SIRPalpha-41 BBL fusion protein, are administered to a subject in need thereof.
A plurality of the anti-cancer agents that may be used with a plurality of specific embodiments of the present invention include, but are not limited to, acitretin; doxorubicin; acodazole hydrochloride; dyclonine; doxorubicin; aldolizhen; aldesleukin; hexamethylmelamine; an Bomei element; amitraz acetate; aminoglutethimide; amsacrine; anastrozole; anthranilate; asparaginase; qu Linjun element; azacitidine; azatepa; dorzolomycin; BAMASITANG; benzotepa; bicalutamide; hydrochloride acid bisantrene; binaford mesylate; the comparison is newer; bleomycin sulfate; sodium buconazole; bromopirimin; busulfan; actinomycin C; carbosterone; carpronium chloride; a card Bei Tim; carboplatin; carmustine; cartubicin hydrochloride; the card is folded for new use; sidefagon; chlorambucil; sirolimus; cisplatin; cladribine; kelinaton mesylate; cyclophosphamide; cytarabine; azazolamide; actinomycin D; n-oxytetracycline hydrochloride; a decitabine; right omaboplatin; dezaguaning; decibutamine mesylate; deaquinone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxib citrate; drotaandrosterone propionate; daptomycin; eda traxas; efluromithine hydrochloride; elsamitrucin; enlobaplatin; enpramine ester; epiridine; epirubicin hydrochloride; erbzol; exenatide hydrochloride; estramustine; estramustine sodium phosphate; itraconazole; etoposide; etoposide phosphate; chloramphenicol; chloramphenicol; chloramphenicol; fadrozole hydrochloride; fazab; fenretinide; fluorouridine; fludarabine phosphate; fluorouracil; fluoxetine; a phosphoquinolone; fosetrexed sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; tamofosin; interferon alpha-2 a; interferon alpha-2 b; interferon alpha-n 1; interferon alpha-n 3; interferon beta-Ia; interferon gamma-Ib; platinum isopropoxide; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprorelin acetate; liazole hydrochloride; lome Qu Suona; lomustine; losoxanone hydrochloride; maxolol; maytansine; nitrogen mustard hydrochloride; megestrol acetate; melengestrol acetate; melphalan; minoxidil; mercaptopurine; methotrexate; methotrexate sodium; chlorphenidine; rituximab; rice Ding Duan; mitomycin; mitomycin; mitoJielin; mi Tuoma stars; mitomycin C; mitoparvalin; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; norramycin; oxaliplatin; an oxy Shu Lun; paclitaxel; pegapase; a pelimycin; pentose mustard; pelargomycin sulfate; pesphosphamide; pipobromide; piposulfan; pyridine Luo Enkun hydrochloride; plicamycin; pralometan; porphin sodium; pofemycin; prednisomustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazolofuranomycin; lipoadenosine; rogestini; sha Fenge; sha Fenge with hydrochloric acid; semustine; xin Quqin; sodium phosphoacetoacetate; rapamycin; germanium spiroamine hydrochloride; spiromustine; spiroplatinum; streptozotocin; chain zoxing; sulfochlorphenylurea; tarithromycin; taxol; sodium tecogalan; tegafur; tilonthraquinone hydrochloride; temopofen; teniposide; luo Xilong; testosterone internal fat; thioazane; thioguanine; thiotepa; thiazole furaline; tirapazamine; topotecan hydrochloride; toremifene citrate; tramadol acetate; troxib phosphate; trimetha sand; triclosan glucuronate; triptorelin; tobrachlorazole hydrochloride; uramustine; uretidine; vaptan; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinblastine sulfate; vinpocetine sulfate; vincristine sulfate; vinorelbine tartrate; vinorelbine sulfate; vinblastidine sulfate; fucloxazole; platinum; clean stastatin; zorubicin hydrochloride. Various additional antineoplastic agents include those disclosed in section 52 of antineoplastic agents (Paul Calabresi and Bruce A. Chabner), and pages 1202-1263 of Goodman and Gilman's "pharmacological basis of therapeutics" 8 th edition, of Magnus-Hill GmbH, 1990.
According to various embodiments, the anti-cancer agent comprises an antibody.
According to various specific embodiments, the antibody is selected from the group consisting of rituximab, cetuximab, trastuzumab, ibritumomab, alemtuzumab, gemtuzumab, temozolomab, panitumumab, belimumab, bevacizumab, bivalizumab-maytansine (Bivatuzumab mertansine), bordetemumab, brenzavib, rituximab-vitamin bestatin (Brentuximab vedotin), cetuximab, daclizumab, adalimumab, bei Zuoluo mab, cetuximab-pekou (Certolizumab pegol), certuzumab-bogus (Citatuzumab bogatox), daritumumab, ding Tuo mab, ibritumomab, erluzumab, etamomab, gemtuzumab-ozagritumomab, ji Tuo, cetuximab, sibutrab, oxuzumab, trastuzumab, rituximab, and rituximab.
According to various embodiments, the antibody is selected from the group consisting of rituximab and cetuximab.
According to various embodiments, the therapeutic agent that is combined with and administered with the compositions of the present invention comprises an anti-inflammatory agent (e.g., antibiotics and antivirals).
According to various embodiments, the therapeutic agent that is combined with and administered to the compositions of the present invention comprises an immunosuppressant (e.g., white blood cell growth hormone (GCSF) and other bone marrow stimulants, steroids).
According to various embodiments, the combination therapy has an additional effect.
According to various embodiments, the combination therapy has a synergistic effect.
According to another aspect of the present invention there is provided an article of manufacture identified for use in treating a disease that may benefit from activating a plurality of immune cells, the article of manufacture comprising: a packaging material containing a therapeutic agent for treating the disease; and a SIRPalpha-41 BBL fusion protein, a polynucleotide encoding the fusion protein, a nucleic acid construct encoding the fusion protein, or a host cell expressing the fusion protein.
According to various embodiments, a therapeutic agent for treating the disease; and a SIRPalpha-41 BBL fusion protein, a polynucleotide encoding the fusion protein, a nucleic acid construct encoding the fusion protein, or a host cell expressing the fusion protein are packaged in separate containers.
According to various embodiments, a therapeutic agent for treating the disease; and a SIRPalpha-41 BBL fusion protein, a polynucleotide encoding the fusion protein, a nucleic acid construct encoding the fusion protein, or a host cell expressing the fusion protein are packaged in a co-formulation.
As used herein, in one embodiment, the term "amino acid derivative" or "derivative" refers to a group derived from a naturally or non-naturally occurring amino acid, as described and illustrated herein. Various amino acid derivatives will be apparent to those skilled in the art and include, but are not limited to, esters of a plurality of naturally and non-naturally occurring amino acids, amino alcohols, amino aldehydes, amino lactones, and N-methyl derivatives. In one embodiment, a derivative of an amino acid is provided as a substituent of a compound described herein, wherein the substituent is-NH-G (Sc) -C (0) -Q or-OC (0) G (S c ) -Q, wherein Q is-SR, -NRR or alkoxy; r is a hydrogen atom or an alkyl group; s is S c A side chain which is a naturally and non-naturally occurring amino acid; g is C 1 -C 2 An alkyl group. At the position ofIn certain embodiments, G is Ci alkyl, and S c Is selected from the group consisting of hydrogen atoms, alkyl groups, heteroalkyl groups, aralkyl groups, and heteroaralkyl groups.
As used herein, in one embodiment, the terms "peptide," "polypeptide," or "protein" are used interchangeably herein to originate from a natural biological source, be it synthetic, or be produced by recombinant techniques. It may be produced by any means known in the art of peptide or protein synthesis, including chemical synthesis. For solid phase peptide synthesis, a summary of many techniques can be found in J.M. Stewart and J.D. Young, solid phase peptide synthesis, W.H. Freeman Co. (San Francisco), 1963 and J.Meienhofer, hormone proteins and peptides, vol.2, p.46, academic Press (New York), 1973. For classical solution synthesis see g.schroder and k.lupke, peptides, vol.1, academic Press (New York), 1965. One or more amino acids may be modified, for example, by the addition of a chemical entity, such as a carbohydrate group, a phosphate group, a farnesyl group, an isoframesyl group, a fatty acid group, an acyl group (e.g., acetyl), a linker for conjugation, functionalization, or other known protecting/blocking groups. The peptide or the protein multiple modification through gene synthesis, site-directed mutagenesis (for example based on PCR method) or random mutation (such as Ethyl Methyl Sulfonate (EMS)), through exonuclease deletion, through chemical modification, or through and encoding a heterologous domain or a binding protein polynucleotide sequence fusion to introduce.
As used herein, in one embodiment, the term "peptide" may be a plurality of fragments, a plurality of derivatives, a plurality of analogs, or a plurality of variants of the foregoing peptides, and any combination thereof. Fragments of the plurality of peptides, as that term or phrase is used herein, include proteolytic fragments as well as deleted fragments. The plurality of variants of the plurality of peptides include fragments having varying amino acid sequences due to amino acid substitutions, deletions or insertions, and peptides.
The plurality of variants may be naturally occurring or non-naturally occurring. Examples include fusion proteins, peptides having one or more residues chemically derivatized by reaction of a functional side group, and peptides comprising one or more naturally occurring amino acid derivatives having twenty standard amino acids. These modifications may also include incorporation of D-amino acids or other non-coding amino acids. In one embodiment, substantially none of the modifications should interfere with the desired biological activity of the peptides and fragments thereof. In another embodiment, a plurality of modifications can alter a characteristic of the peptide and fragments thereof, such as stability or half-life, without interfering with the desired biological activity of the peptide and fragments thereof. In one embodiment, the terms "peptide" or "protein" as used herein may be used interchangeably with the same meaning and properties.
In one embodiment, to facilitate recovery, the expressed coding sequence may be engineered to encode the peptides of the invention and fused to a cleavable moiety. In one embodiment, a fusion protein can be designed such that the peptide can be easily isolated by affinity chromatography; for example by being fixed to a tubular string specific for said cleavable moiety. In one embodiment, a cleavage site is engineered between the peptide and the cleavable moiety, and the peptide can be released from the chromatographic column by treatment with a suitable enzyme or reagent that specifically cleaves the fusion protein at this site [ e.g., see Booth et al, immunol. Lett.19:65-70 (1988); and Gardella et al, J.biol. Chem.265:15854-15859 (1990) ].
In one embodiment, each of the peptides forming the fusion proteins of the invention (also referred to herein as "the peptides") may also be synthesized using multiple expression systems in vitro. In one embodiment, multiple synthetic methods in vitro are well known in the art, and multiple compositions of the system are commercially available.
In one embodiment, a peptide of the invention can be produced using recombinant DNA techniques. A "recombinant" peptide or protein refers to a peptide or protein produced by recombinant DNA techniques; that is, by cells transformed with an exogenous DNA construct encoding the peptide or protein.
Thus, according to another aspect of the invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding any of the fusion proteins described above.
According to specific embodiments, the polynucleotide comprises SEQ ID NO. 8.
According to various specific embodiments, the polynucleotide consists of SEQ ID NO. 8.
According to various specific embodiments, the polynucleotide is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the nucleic acid sequence set forth in SEQ ID No. 8.
As used herein, the term "polynucleotide" refers to a single-or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and/or a complex polynucleotide sequence (e.g., a combination of the foregoing).
In order to express exogenous SIRPalpha-41 BBL in a plurality of mammalian cells, a polynucleotide sequence encoding the SIRPalpha-41 BBL is preferably ligated to a nucleic acid construct suitable for expression in mammalian cells. Such nucleic acid constructs comprise a promoter sequence which directs transcription of the polynucleotide in the cell in a constitutive or inducible manner.
Thus, according to various embodiments, there is provided a nucleic acid construct comprising the polynucleotide and a regulatory element for directing expression of the polynucleotide in a host cell.
The nucleic acid constructs of some embodiments of the invention (also referred to herein as a "expression vector") include a plurality of additional sequences that render such vectors suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may also contain a transcription and translation initiation sequence, a transcription and translation termination sequence, and a polyadenylation signal. By way of example, such constructs typically include a 5-terminal long repeat (5 'LTR), a tRNA binding site, a packaging signal, a start point for second strand DNA synthesis, and a 3-terminal long repeat (3' LTR) or portion thereof.
The nucleic acid constructs of some embodiments of the invention generally comprise a signal sequence for secretion of a peptide from a host cell in which they are located. Preferably, the signal sequence used for this purpose is a mammalian signal sequence or a signal sequence of the polypeptide variant of some embodiments of the invention.
Eukaryotic promoters typically contain two types of recognition sequences, a TATA box and an upstream promoter element. The TATA box located 25 to 30 base pairs upstream of the transcription initiation site is thought to be involved in directing RNA polymerase to begin RNA synthesis. Other upstream promoter elements determine the initiation rate of transcription.
Preferably, the promoters utilized by the nucleic acid constructs of some embodiments of the invention are active in the particular cell population being transformed. Examples of promoters for specific cell types and/or tissue types, such as liver-specific albumin [ Pinker et al, (1987) Genes Dev.1:268-277], lymphoid-specific promoters [ Calame et al, (1988) adv. Immunol.43:235-275]; in particular the T Cell receptor [ Winito et al, (1989) EMBO J.8:729-733] and the immunoglobulin [ Banerji et al, (1983) Cell 33729-740 ]; neural specific promoters, such as the neurofilament promoter [ Byrne et al (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas specific promoters [ Edlunch et al (1985) Science 230:912-916], or mammary gland specific promoters, such as the whey protein promoter (U.S. Pat. No. 4,873,316 and European patent publication No. 264,166).
Multiple enhancer elements can be stimulated from linked homologous or heterologous promoters to carry out transcription up to 1000-fold. The enhancer is active when it is placed downstream or upstream of the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, SV40 early gene enhancers are suitable for many cell types. Other enhancer/promoter combinations suitable for some embodiments of the invention include those derived from polyomavirus, human or murine Cytomegalovirus (CMV), terminal long repeats from various retroviruses, such as murine leukemia virus, murine or rous sarcoma virus, and HIV. See enhancers and eukaryotic expression, cold Spring Harbor Press, cold Spring Harbor, n.y.1983, incorporated herein by reference.
In the construction of expression vectors, the promoter is preferably located at about the same distance as the heterologous transcription start site, as it would be in its natural condition transcription start site. As is known in the art, however, some variation in this distance can be accommodated without losing the function of the promoter.
Multiple polyadenylation sequences may also be added to the expression vector in order to increase the translation efficiency of sirpa-41 BBL mRNA. Accurate and efficient polyadenylation requires two different sequence elements: a GU or U-rich sequence downstream of the polyadenylation site, and a highly conserved 6 nucleotide sequence AAUAAA located 11 to 30 nucleotides upstream. A plurality of termination signals suitable for use in some embodiments of the invention include those derived from SV 40.
In addition to the elements already described, the expression vector of some embodiments of the invention may generally contain other specific elements aimed at increasing the degree of expression of the cloned nucleic acid or at facilitating the identification of a plurality of cells carrying the recombinant DNA. For example, some animal viruses contain multiple DNA sequences for promoting extrachromosomal replication of the viral genome in multiple permissive cell (permissive cell) types. Multiple plastids tolerating these viral replicons undergo episomal replication as long as multiple appropriate factors are provided by multiple genes carried on the plastid or multiple genes with the genome of the host cell.
The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, the vector is then amplified in eukaryotic cells by a plurality of suitable selectable markers. If the vector does not contain a eukaryotic replicon, no episomal amplification is possible. Alternatively, the recombinant DNA is integrated into the genome of the engineered cell, where the promoter directs the expression of the desired nucleic acid.
The expression vector of some embodiments of the invention may further include additional polynucleotide sequences, e.g., sequences that allow translation of proteins from a single mRNA, such as an Internal Ribosome Entry Site (IRES), and sequences for genomic integration of promoter-chimeric polypeptides.
It should be understood that the various elements in the expression vector may be arranged in a variety of configurations. For example, enhancer elements, promoters, etc. may be present in a reverse complement or in a complementary arrangement, such as an antiparallel strand, and even the polynucleotide sequence(s) encoding a sirpa-41 BBL may be arranged in a "head-to-tail" arrangement. While such various configurations are more likely to occur for multiple non-coding elements of the expression vector, multiple alternative configurations of coding sequences within the expression vector are also contemplated.
Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2 (+/-), pSecTag2, pDISPLAY, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, available from the England Biotechnology; pCI available from Bomeka (Promega); pMbac, pPbac, pBK-RSV and pBK-CMV obtainable from the history Cui Te gene (Strategene); pTRES obtained from cloning technology (Clontech), and derivatives thereof.
A plurality of expression vectors containing regulatory elements derived from eukaryotic viruses such as retroviruses may also be used. SV40 vectors include pSVT7 and pMT2. The plurality of vectors derived from bovine papilloma virus include pBV-1MTHA, and the plurality of vectors derived from human herpesvirus include pHEBO and p2O5. Other exemplary vectors include pMSG, pav009/a+, pMTO10/a+, pmarneo-5, baculovirus pDSVE, and other vectors that allow expression of proteins under the direction of SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, rous sarcoma virus promoter, polyhedrin promoter, or other promoters that exhibit efficient expression in eukaryotic cells.
As described above, in many instances, viruses are very specialized infectious agents that have evolved multiple defense mechanisms to evade the host. Typically, viruses infect and propagate in a number of specific cell types. The targeting specificity of viral vectors exploits its natural specificity to specifically target a plurality of predetermined cell types, thereby introducing a recombinant gene into infected cells. Thus, the type of vector used in some embodiments of the invention will depend on the cell type to be transformed. The ability to select an appropriate vector according to the cell type to be transformed is well within the ability of the ordinarily skilled artisan, and thus a general description of such selection considerations is not provided herein. For example, bone marrow cells can be targeted by using human T-cell leukemia virus type I (HTLV-I) and kidney cells can be targeted by using a heterologous promoter present in the baculovirus class of Autographa californica nuclear polyhedrosis virus (AcMNPV), as described in Liang CY et al, 2004 (Arch virol. 149:51-60).
A variety of recombinant viral vectors are helpful for the expression of SIRPalpha-41 BBL in vivo because they offer many advantages, such as lateral infection and target specificity. The lateral infection is inherent in, for example, the life cycle of a retrovirus, and is a process by which a single infected cell produces many budding (budd) progeny virions and infects multiple adjacent cells. The result is a large area that is rapidly infected, with a large portion of the area not being initially infected by the original virus particle. This is in contrast to a vertical type of infection, in which the infectious agent is transmitted only through maternal progeny. The plurality of viral vectors produced are also not capable of lateral transmission. Such a property is helpful if the desired purpose is to introduce a specific gene into only a localized number of target cells.
Various methods may be used to introduce the expression of some embodiments of the invention in vivo into a variety of cells. Such methods are generally described in Sambrook et al, molecular cloning: laboratory manuals, cold Springs Harbor Laboratory, new York (1989, 1992), ausubel et al, methods of contemporary molecular biology, john Wiley and Sons, baltimore, md. (1989), chang et al, somatic gene therapy, CRC Press, ann Arbor, mich. (1995), vega et al, gene targeting, CRC Press, ann Arbor Mich. (1995), vector: molecular cloning vectors and their general overview of use are described in Butterworth, boston Mass. (1988) and Gilboa et at. [ Biotechniques 4 (6): 504-512,1986], and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, for positive-negative selection methods, see U.S. Pat. nos. 5,464,764 and 5,487,992.
The introduction of nucleic acids by viral infection provides several advantages over other methods such as liposome transfection and electroporation, since higher transfection efficiencies can be obtained due to the infectious nature of the virus.
Currently, a number of preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, herpes simplex virus type I, or adeno-associated virus (AAV) and lipid-based systems. A number of useful lipids for lipid-mediated transfer of genes are, for example, DOTMA, DOPE and DC-Chol [ Tonkinson et al Cancer Investigation,14 (1): 54-65 (1996) ]. A number of more preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, adeno-associated viruses, lentiviruses or retroviruses. A viral construct, such as a retroviral construct, includes at least one transcriptional promoter/enhancer or locus definition unit(s), or other elements that control gene expression by other means, such as alternative splicing, nuclear RNA export, or post-translational modification of the message. Such vector constructs also include a packaging signal, terminal long repeats (LTRs) or a portion thereof, and multiple positive and negative strand primer binding sites appropriate for the virus being used, unless it is already present in the viral vector. In addition, such constructs typically include a signal sequence for secretion of the peptide from a host cell in which they are located. Preferably, the signal sequence used for this purpose is a mammalian signal sequence or a variant of the polypeptide of some embodiments of the invention. Alternatively, the construct may also comprise a signal directing polyadenylation, and one or more restriction sites and translation termination sequences. By way of example, such constructs will typically include a 5'LTR, a tRNA binding site, a packaging signal, a start point for the synthesis of the second strand of DNA, and a 3' LTR or portion thereof. Other carriers that may be used are non-viral, such as cationic lipids, polylysine, and dendrimers.
As noted above, in addition to containing the necessary elements for transcription and translation of the inserted coding sequence, the expression constructs of some embodiments of the invention may also include sequences engineered to enhance stability, manufacture, purity, yield, or toxicity of the expressed peptide. For example, the expression of a fusion protein or a cleavable fusion protein comprising the sirpa-41 BBL fusion protein and a heterologous protein of some embodiments of the invention may be engineered. Such fusion proteins can be designed so that they can be easily isolated by affinity chromatography; for example by being immobilized on a column specific for said heterologous protein. Wherein a cleavage site is engineered between the SIRPalpha-41 BBL and the heterologous protein, and the SIRPalpha-41 BBL is released from the chromatography column by treatment with a suitable enzyme or reagent that disrupts the cleavage site [ e.g., see Booth et al (1988) Immunol. Lett.19:65-70; gardella et al, (1990) J.biol.chem.265:15854-15859].
The invention also contemplates a plurality of cells comprising the compositions described herein.
Thus, according to particular embodiments, a host cell is provided comprising the sirpa-41 BBL fusion protein, the polynucleotide encoding the sirpa-41 BBL fusion protein, or the nucleic acid construct encoding the sirpa-41 BBL fusion protein.
As mentioned above, various prokaryotic or eukaryotic cells may be used as a plurality of host expression systems in order to express a plurality of the polypeptides of some embodiments of the invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant phage DNA, plastid DNA, or cosmid DNA expression vector comprising a coding sequence, yeasts transformed with a plurality of recombinant yeast expression vectors comprising a coding sequence; a plurality of plant cell systems infected with a plurality of recombinant viral expression vectors (e.g., cauliflower mosaic virus, caMV; tobacco mosaic virus, TMV) or transformed with recombinant plastid expression vectors comprising a coding sequence, e.g., ti plastids. Mammalian expression systems may also be used to express a plurality of the polypeptides of some embodiments of the invention.
Examples of bacterial constructs include E.coli expression vectors of the various pET families (Studier et al (1990) Methods in enzymol.185:60-89).
Various examples of eukaryotic cells that may be used with the teachings of the present invention include, but are not limited to, mammalian cells, fungal cells, yeast cells, insect cells, algal cells, or plant cells.
In yeast, multiple vectors containing constitutive or inducible promoters may be used, as disclosed in U.S. patent application publication No. 5,932,447. Alternatively, multiple vectors that facilitate integration of multiple foreign DNA sequences into the yeast chromosome may be used.
In the case of plant expression vectors, expression of the coding sequence may be driven by a number of promoters. For example, a variety of viral promoters may be used, such as the 35S RNA and 19S RNA promoters of CaMV [ Brisson et al (1984) Nature 310:511-514], or the coat protein promoter of TMV [ Takamatsu et al (1987) EMBO J.6:307-311]. Alternatively, a variety of plant promoters may be used, such as the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RUBISCO) [ Coruzzi et al (1984) EMBO J.3:1671-1680 and Brogli et al, (1984) Science 224:838-843], or heat shock promoters, such as soybean hsp17.5-E or hsp17.3-B [ Gurley et al (1986) mol.cell.biol.6:559-565]. These constructs can be introduced into a plurality of plant cells by using Ti plastids, ri plastids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and other techniques known to the skilled artisan. See, e.g., weissbach & Weissbach,1988, methods for plant molecular biology, academic Press, NY, section VIII, pp 421-463.
Other expression systems, such as insect or mammalian host cell systems, which are well known in the art, may also be used in some embodiments of the present invention.
According to various embodiments, the cell is a mammalian cell.
According to various specific embodiments, the cell is a human cell.
According to a specific embodiment, the cell is a cell line.
According to another specific embodiment, the cell is a primary cell.
The cells may be derived from a suitable tissue including, but not limited to, blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair, skin, bone, breast, uterus, bladder, spinal cord, or various bodily fluids. The cells may be derived from any stage of development, including embryonic, fetal and adult stages; and developmental origin, i.e. ectodermal, mesodermal and endodermal origin.
A number of non-limiting examples of various mammalian cells include: monkey kidney CV1 strain transformed with SV40 (COS, e.g., COS-7, ATCC CRL 1651); human embryonic kidney lines (HEK 293 or subclones of HEK293 cells for growth in suspension culture, graham et al, j.gen virol.,36:59 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse cetrorelix cells (TM 4, mather, biol. Reprod.,23:243-251 1980); monkey kidney cells (CV 1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HeLa, ATCC CCL 2); NIH3T3, jurkat, canine kidney cells (MDCK, ATCC CCL 34); brulo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562,ATCC CCL51); TRI cells (Mather et al, annals N.Y. Acad.Sci.,383:44-68 1982); human fetal lung fibroblasts (MRC 5); FS4 cells; and human liver tumor lines (Hep G2), per.c6, human chronic myelogenous leukemia cells (K562), and chinese hamster ovary Cells (CHO).
According to some embodiments of the invention, the mammalian cell is selected from the group consisting of chinese hamster ovary Cells (CHO), HEK293, per.c6, human fibrosarcoma cells (HT 1080), mouse myeloma cells (NS 0), mouse myeloma cells (SP 2/0), BHK, human leukemia cells (Namalwa), COS, heLa and Vero cells.
According to some embodiments of the invention, the host cells comprise chinese hamster ovary Cells (CHO), per.c6 and 293 (e.g., expi 293F) cells.
According to another aspect of the invention there is provided a method for making a sirpa-41 BBL fusion protein, the method comprising: the polynucleotides and the nucleic acid constructs as described herein are expressed in a host cell.
According to a number of specific embodiments, the method comprises: isolating the fusion protein.
According to various embodiments, recovery of the recombinant polypeptide is performed after a suitable period of incubation. The phrase "recovery of recombinant polypeptide" refers to collection of the entire fermentation medium containing the polypeptide, and does not necessarily involve additional isolation or purification steps. Nonetheless, the plurality of polypeptides of some embodiments of the invention can be purified by using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, colloidal filtration chromatography, reverse chromatography, concanavalin A chromatography, mixed mode chromatography, metal affinity chromatography, lectin affinity chromatography, chromatofocusing, and differential dissolution.
In some embodiments, the plurality of recombinant peptides, fragments thereof, or plurality of peptides are synthesized and purified; their therapeutic efficacy can be measured in vivo or in vitro. In one embodiment, the plurality of recombinant fragments or plurality of peptides of the invention can be determined using a variety of assays, including cellular activity, survival of genetically transformed mice, and expression of megakaryocyte and lymphoid RNA markers.
In one embodiment, a peptide of the invention comprises at least 3 amino acids. In another embodiment, a peptide comprises at least 5 amino acids. In another embodiment, a peptide comprises at least 10 amino acids. In another embodiment, a peptide comprises at least 20 amino acids. In another embodiment, a peptide comprises at least 25 amino acids. In other embodiments, a peptide comprises at least 30 amino acids or at least 50 amino acids or 75 amino acids, or 100 amino acids, or 150 amino acids, or 200 amino acids, or 250 amino acids or 300 amino acids or 350 amino acids or 400 amino acids. In one embodiment, a peptide of the invention consists essentially of at least 5 amino acids. In another embodiment, a peptide consists essentially of at least 10 amino acids. In other embodiments, a peptide consists essentially of at least 30 amino acids or at least 50 amino acids or 75 amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids, or 200 amino acids, or 250 amino acids, or 300 amino acids, or 350 amino acids, or 400 amino acids. In one embodiment, a peptide of the invention consists of at least 5 amino acids. In another embodiment, a peptide consists of at least 10 amino acids. In other embodiments, a peptide consists of at least 30 amino acids or at least 50 amino acids or 75 amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids, or 200 amino acids, or 250 amino acids or 300 amino acids or 350 amino acids or 400 amino acids or 500 or 600 or 700 amino acids.
As used herein, the terms "peptide" and "fragment" are used interchangeably in one embodiment to have exactly the same meaning and properties. As used herein, in one embodiment, the term "peptide" includes natural peptides (degradation products, synthetic peptides or recombinant peptides) and peptidomimetics (typically synthetic peptides), such as peptide-like and semi-peptide-like peptides of various peptide analogs, which may have, for example, modifications to make the peptide more stable or capable of penetrating into bacterial cells in vivo. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, including, but not limited to, CH2-NH, CH2-S, CH 2-s= O, O =c-NH, CH2-O, CH2-CH2, s=c-NH, ch=ch or cf=ch, backbone modifications, and residue modifications. Various methods for preparing peptidomimetic compounds are well known in the art and are specifically described in quantitative drug design, c.a. ramsden Gd., section 17.2,F.Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.
The peptide bond (-CO-NH-) in the peptide may be substituted, for example, by N-methylation bond (-N (CH 3) -CO-), ester bond (-C (R) H-C-O-O-C (R) -N-), ketomethylene bond (-CO-CH 2-), alpha-aza (aza) bond (-NH-N (R) -CO-), where R is any alkyl group, such as methyl, kappa (carb) bond (-CH 2-NH-), hydroxy vinyl bond (-CH (OH) -CH 2-), thioamide bond (-CS-NH-), olefinic double bond (-CH=CH-), anti-amide bond (-NH-CO-), peptide derivative (-N (R) -CH 2-CO-), where R is a "normal" side chain, naturally occurring on a carbon atom.
These modifications can occur at any bond on the peptide chain, and even at the same time at several (2 to 3) bonds.
Natural aromatic amino acids, tryptophane (Trp), tyrosol (Tyr) and phenylalanine (Phe), may be substituted for synthetic unnatural acids such as TIC, naphthylalanine (Nol), cyclomethylated derivatives of phenylalanine, halogenated derivatives of phenylalanine or o-methyl-tyramine.
In one embodiment, the peptides of the invention further comprise a detectable tag. As used herein, in one embodiment, the term "detectable label" refers to any portion that can be detected by a person using techniques known in the art. The detectable tag used in the multiple screening methods of the invention can be multiple peptide sequences. Alternatively, the detectable label may be removed by a plurality of chemical reagents or a plurality of enzymatic methods, such as protein hydrolysis. For example, the term "detectable tag" includes chitin binding protein (CBD) -tag, maltose Binding Protein (MBP) -tag, glutathione S-transferase (GST) -tag, histidine (His) -tag, FLAG tag, epitope tag, such as V5-tag, c-myc-tag, and hemagglutinin-tag, and fluorescent tag, such as Green Fluorescent Protein (GFP), red Fluorescent Protein (RFP), yellow Fluorescent Protein (YFP), blue Fluorescent Protein (BFP), and Cyan Fluorescent Protein (CFP); and derivatives of these tags, or any tags known in the art. The term "detectable label" may also include the term "detectable label".
In one embodiment, a peptide of the invention is an isolatable peptide. Such an isolatable peptide may comprise a peptide-tag.
The plurality of peptides of some embodiments of the invention are preferably used in a linear form, but it should be understood that the cyclic form of the peptides may also be used without severely interfering with the peptide characteristics by cyclization.
As used herein, in one embodiment, the term "amino acid" refers to naturally occurring and synthetic alpha, beta, gamma, or delta amino acids, and includes, but is not limited to, amino acids found in proteins, i.e., glycine, alanine, valine, leucine, isophthaline, methionine, phenylalanine, tryptophane, proline, serine, threine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. In certain embodiments, the amino acid is in the L configuration. Alternatively, the amino acid may be a derivative of alanyl, valyl, white acyl, iso-white acyl, prolyl, amphetaminyl, tryptophanyl, methionyl, glycyl, seryl, threonyl, cysteinyl, tyrosyl, asparaginyl, glutaminyl, asparaginyl, glutamyl, lysyl, arginyl, histidinyl, β -alanyl, β -valyl, β -white acyl, β -iso-white acyl, β -prolyl, β -amphetaminyl, β -tryptophanyl, β -methionyl, β -glycyl, β -seryl, β -Su Anxian, β -cysteinyl, β -tyrosyl, β -asparaginyl, β -glutaminyl, β -asparaginyl, β -glutamyl, β -Lai Anxian, β -arginyl, β -histamine.
Because the peptides of the invention are preferably used in therapeutic or diagnostic applications where the peptides need to be in a soluble form, the peptides of some embodiments of the invention preferably include one or more amino acids of unnatural or natural polarity, including but not limited to serine and threonine, which can increase the solubility of the peptide due to their hydroxyl-containing side chains.
As used herein, in one embodiment, the phrase "conservatively modified variants" applies to both amino acid sequences and nucleic acid sequences. "amino acid variant" refers to an amino acid sequence. Conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence as essentially identical or related (e.g., naturally occurring close) sequences, relative to the sequence of a particular nucleic acid. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode a large portion of the protein. For example, codons GCA, GCC, GCG and GCU both encode the amino acid alanine. Thus, at each position where a codon is designated as alanine, the codon can be changed to another corresponding codon as described without changing the encoded polypeptide. Such nucleic acid variants are "silent variants," which are one of the conservatively modified variants. Each nucleic acid sequence encoding a peptide herein also describes a plurality of silencing variants of the nucleic acid. The skilled artisan will appreciate that in some cases, each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, silent variants of a nucleic acid which encodes a polypeptide are inherent in a described sequence relative to the expression product.
With respect to amino acid sequences, the skilled artisan will appreciate that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a low proportion of amino acids in the coding sequence are "conservatively modified variants" that include such alterations resulting in the substitution of an amino acid with a chemically similar amino acid. Conservative representations of amino acids that provide functional similarity are well known in the art. Guidance as to which amino acid changes are likely to be phenotypically silent can also be found in Bowie et al 1990,Science 247:1306 1310. Furthermore, such conservatively modified variants do not exclude polymorphic variants, interspecies homologs, and alleles. Typical conservative substitutions include, but are not limited to: 1) Alanine (a), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Iso-leucine (I), leucine (L), methionine (M), valine (V); 6) Amphetamine acid (F), tyrosine (Y), tryptophane acid (W); 7) Serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., cright on, protein (1984)). Amino acids may be substituted based on properties associated with the side chains, e.g., amino acids having polar side chains may be substituted, such as serine (S) and threonine (T); amino acids based on the charge of a side chain, such as arginine (R) and histidine (H); and amino acids having hydrophobic side chains, such as valinic acid (V) and leucine (L). As indicated, the plurality of alterations are typically of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein.
Chemical modification of proteins
In the present invention, any portion of a protein of the present invention may optionally be chemically modified, i.e., altered by the addition of multiple functional groups. For example, amino acid side residues that occur in a natural sequence may optionally be modified, although as described below, other portions of the protein may alternatively be modified in addition to or instead of the amino acid side residues. For example, if a chemically modified amino acid is added after a chemical synthesis process, the modification may optionally be performed during synthesis of the molecule. However, chemical modification of an amino acid is also possible when the amino acid is already present in the molecule ("in situ" modification).
The amino acids of any sequence region of the molecule may be optionally modified according to any of the following various exemplary modification types (in peptides that are conceptually considered "chemically modified"). Non-limiting exemplary types of modification include carboxymethylation, acylation, phosphorylation, glycosylation, or fatty acylation. An ether linkage may alternatively be used to link the hydroxyl group of serine or threonine to the hydroxyl group of a saccharide. The amide bond is optionally used to link the carboxyl group of glutamic acid or aspartic acid to a monoamine group on a saccharide (Garg and Jeanloz, progress of carbohydrate chemistry and biochemistry, vol.43, academic Press (1985); kunz, ang. Chem. Int. Ed. Englist 26:294-308 (1987)). Acetal and ketal bonds can also optionally be formed between amino acids and carbohydrates. Fatty acid acyl derivatives can alternatively be prepared, for example, by acylation of a free amine group (e.g., lysine) (Toth et al, peptides: chemistry, structure and biology, rivier and Marshal, eds., ESCOM publication, leiden,1078-1079 (1990)).
As used herein, the term "chemical modification" when referring to a protein or peptide according to the present invention refers to a protein or peptide in which at least one of its amino acid residues is modified by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques well known in the art. A number of examples of many known modifications generally include, but are not limited to: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, formation of a glycophospholitidyl inositol (GPI) anchor, covalent attachment of a lipid or lipid derivative, methylation, tetradecylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process.
Other types of modifications optionally include the addition of a cycloalkane moiety to a biomolecule, such as a protein, as described in PCT application No. WO 2006/050262, which is incorporated herein by reference as if fully set forth herein. These moieties are designed for use with multiple biomolecules and optionally to confer various properties to the protein.
Furthermore, modifications may be made at any site on a protein. For example, the pegylation may optionally be performed on a glycosylated portion of a protein, as described in PCT application No. WO 2006/050247, which is incorporated by reference as if fully set forth herein. One or more polyethylene glycol (PEG) groups may optionally be added to O-linked and/or N-linked glycosylation. The PEG group may alternatively be branched or linear. Alternatively, any type of water-soluble polymer may be attached to a protein at a glycosylation site via a glycosyl linkage.
"PEGylated protein" refers to a protein, or fragment thereof, having biological activity, having a polyethylene glycol (PEG) moiety covalently bound to an amino acid residue of the protein.
"polyethylene glycol" or "PEG" refers to a polyalkylene glycol compound or derivative thereof, with or without multiple coupling agents or derivatization with coupling or active moieties (e.g., moieties with thiols, triflates, aziridines, ethylene oxide, or preferably with a maleimide). Compounds such as maleimide-based monomethoxy PEG are exemplary or activated PEG compounds of the present invention. Other polyalkylene glycol compounds, such as polypropylene glycol, may be used in the present invention. Other suitable polyalkylene glycol compounds include, but are not limited to, charged or neutral polymers of the following types: dextran, polysialic acid or other carbohydrate-based polymers, amino acid polymers, and biotin derivatives.
Altered glycosylated protein modifications
The proteins of the invention may be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used herein, "altered" means having one or more removed carbohydrates, and/or having at least one glycosylation site added to the original protein.
Glycosylation of proteins is typically either N-linked or O-linked. N-linked refers to attaching the carbohydrate moiety to a side chain having an asparagine residue. A plurality of tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid other than proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, any of these tripeptide sequences that are present in a polypeptide may result in a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylglucosamine, galactose or xylose to a monohydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
The addition of multiple glycosylation sites to the proteins of the invention can be conveniently accomplished by altering the amino acid sequence of the protein, such that the protein contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The alteration may also be achieved by adding or replacing one or more serine or threonine residues in the sequence of the original protein (for O-linked glycosylation sites). The amino acid sequence of the protein may also be altered by introducing changes at the DNA level.
Another way to add multiple carbohydrate moieties to multiple proteins is through chemical or enzymatic coupling of multiple glycosides to multiple amino acid residues of the proteins. Depending on the coupling mode used, a plurality of saccharides may be attached to the amide groups of (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as cysteine, (d) free hydroxyl groups, such as serine, threonine or hydroxyproline, (e) aromatic residues, such as phenylalanine, tyrosine or tryptophan, or (f) glutamine. These methods are described in International application No. WO 87/05330, and Aplin and Wriston, CRC crit.Rev.biochem.,22:259-306 (1981).
Removal of any carbohydrate moiety present on the plurality of proteins of the invention may be accomplished chemically, enzymatically, or by introducing changes in DNA levels. Chemical deglycosylation requires exposing the protein to trifluoromethanesulfonic acid or an equivalent compound. This treatment results in cleavage of most or all of the saccharides except the linked saccharide (N-acetylglucosamine or N-acetylgalactosamine), leaving behind the complete amino acid sequence.
Chemical deglycosylation is described in hakimudin et al, arch. Biochem. Biophys, 259:52 (1987); and Edge et al, anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrates on multiple proteins can be achieved by using a variety of endo-and exo-glycosidases, as described in Thotakura et al, meth. Enzymol, 138:350 (1987).
Pharmaceutical composition
Compositions of some embodiments of the invention (e.g., sirpa-41 BBL fusion proteins, polynucleotides encoding the sirpa-41 BBL fusion proteins, nucleic acid constructs encoding the sirpa-41 BBL fusion proteins, and/or cells) can be provided to an organism itself, or mixed with suitable carriers or excipients in a pharmaceutical composition.
In some embodiments, the invention features a pharmaceutical composition that includes a therapeutically effective amount of a therapeutic agent according to the invention. According to the invention, the therapeutic agent may be a polypeptide as described herein. The pharmaceutical composition according to the invention is further used for the treatment of cancer or an immune related disease as described herein. The therapeutic agents of the invention may be provided to the subject alone or as part of a pharmaceutical composition in which they are admixed with a pharmaceutically acceptable carrier.
As used herein, a "pharmaceutical composition" refers to the preparation of one or more of the active ingredients described herein together with other chemical ingredients, such as physiologically suitable carriers and excipients. A pharmaceutical composition is intended to facilitate the administration of a compound to a living organism.
As used herein, the term "active ingredient" refers to a composition that is responsive to a biological effect (e.g., a SIRPalpha-41 BBL fusion protein, polynucleotide, nucleic acid construct and/or cell as described herein).
As used herein, the term "excipient" refers to an inert substance that is added to a pharmaceutical composition to further facilitate the administration of an active ingredient. Examples of such excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of various starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably to refer to a carrier or a diluent that does not cause significant irritation to an organism and does not negate the biological activity and properties of the compound being administered. These phrases include an adjuvant.
As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Depending on the route of administration, examples of such salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous-containing acids, and the like, and non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxy aromatic acids, N-methyl-and alkali-metal diamines, and the like, and non-toxic organic amines such as N, N '-methyl-and alkali-metal diamines, N' -methyl-and alkali-metal amines, and the like.
According to at least some embodiments of the present invention, a pharmaceutical composition may also include a pharmaceutically acceptable antioxidant. Examples of such pharmaceutically acceptable antioxidants include: (1) Water-soluble antioxidants such as ascorbic acid, cysteamine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) Oil-soluble antioxidants such as ascorbyl palmitate, butyl Hydroxy Anisole (BHA), dibutyl hydroxy toluene (BHT), lecithin, propyl gallate, alpha-vitamin E, and the like; and (3) metal chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. According to at least some embodiments of the present invention, a pharmaceutical composition may also include additives such as detergents and solubilizing agents (e.g., polysorbate-20 (TWEEN 20), polysorbate-80 (TWEEN 80)) and preservatives (e.g., thimerosal, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).
Examples of suitable water-soluble or non-water-soluble carriers that may be applied to the plurality of pharmaceutical compositions according to at least some embodiments of the present invention include: water, buffered saline of various buffer levels (e.g., tris-HCl, acetate, phosphate), pH and ionic strength, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, e.g., olive oil, and injectable organic esters, e.g., ethyl oleate.
Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions may also contain a plurality of adjuvants, such as preserving, wetting, emulsifying and dispersing agents. By the sterilization procedure above, and by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like, the presence of microorganisms can be ensured. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional medium or agent is incompatible with the active compound, its use in a plurality of such pharmaceutical compositions in accordance with at least some embodiments of the present invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.
Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many instances, it is preferred to include an isotonic agent, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of a plurality of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject to be treated and the particular mode of administration. The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form is typically that amount of the composition that produces a therapeutic effect. Typically, this amount will range from about 0.01 to 99 percent of the active ingredient, preferably from about 0.1 to 70 percent, and most preferably from 1 to 30 percent of the active ingredient in combination with a pharmaceutically acceptable carrier, in 100 percent.
Multiple dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus (bolus) may be administered, multiple separate doses may be administered over time, or the doses may be proportionally reduced or increased depending on the degree of urgency of the treatment regimen. It is particularly advantageous to formulate compositions for non-oral administration in dosage unit form for ease of administration and uniformity of dosage. As used herein, the term dosage unit form refers to a plurality of physically discrete units suitable as unitary dosages for administration to a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect with the required pharmaceutical carrier. According to at least some embodiments of the present invention, a specification for the form of the dosage unit is specified and directly depends on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of mixing with such active compounds for the treatment of sensitivity in individuals.
Various techniques for formulating and administering pharmaceuticals can be found in the latest version of the pharmaceutical science of "leimington," Mack Publishing co., easton, PA, which is incorporated herein by reference.
The various pharmaceutical compositions of some embodiments of the present invention may be prepared by various procedures well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
A composition of the invention may be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending on the desired outcome. According to at least some embodiments of the present invention, preferred routes of administration for therapeutic agents include: intravascular delivery (e.g., injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, enteral, rectal, pulmonary (e.g., inhalation), nasal, topical (including transdermal, buccal and sublingual), intravesical, intraocular, intraperitoneal, vaginal, brain delivery (e.g., intraventricular, intracerebral and convection-enhanced diffusion), central Nervous System (CNS) delivery (e.g., intrathecal, peri-medullary and intravertebral) or parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous (IV) and intradermal), transdermal (passive or using iontophoresis or electroporation), transmucosal (e.g., sublingual, nasal, vaginal, rectal or sublingual), or by an implant, or other parenteral route of administration, such as by injection or infusion, or other delivery routes and/or forms of administration known in the art. The phrase "parenteral administration" as used herein means a variety of modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous (intraarticular), intra-articular, subcapsular, subarachnoid, intravertebral, epidural and intrasternal injection and infusion, or use of bioerodible inserts, and may be formulated into dosage forms suitable for each route of administration. In a specific embodiment, a therapeutic agent or a pharmaceutical composition may be administered intraperitoneally or intravenously in accordance with at least some embodiments of the present invention.
When delivered as an aerosol or as a plurality of spray-dried particles having a aerodynamic diameter of less than about 5 microns, the compositions of the present invention may be delivered to the lungs upon inhalation and through the lung epithelial lining to the blood stream. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products may be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the eurChart (Ultravent) nebulizer (Mallinckrodt Inc., st. Louis, mo.); sub-air II (Acorn II) nebulizer (maxster medical manufacture (Marquest Medical Products), englewiod, colo.); pantiline (Ventolin) scale dose inhaler (Glaxo inc.), research Triangle Park, n.c.); and Shi Binhei luxer (Spinhaler) powder inhalers (non-liter corporation (fishens corp.), bedford, mass.). Nektar, alkermes, and Mannkind have approved or in clinical trials inhalable insulin powder formulations, wherein the techniques may be applied to a variety of formulations described herein.
In some in vivo methods, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" means a dose sufficient to treat, inhibit or ameliorate one or more symptoms of the disease to be treated or otherwise provide a desired pharmacological and/or physiological effect. The precise dosage will vary depending on various factors, such as variability associated with the subject (e.g., age, immune system health, etc.), disease, and the treatment being administered. For the polypeptide compositions disclosed herein, the polynucleotides encoding the polypeptides and the nucleic acid constructs and the cells as described herein will present information about a plurality of appropriate dosage levels for treating various symptoms in various patients as further studies are carried out, and one of ordinary skill in the art will be able to determine the appropriate dosage taking into account the therapeutic context, age and general health of the recipient. The selected dosage depends on the desired therapeutic effect on the route of administration and on the duration of the desired treatment. For polypeptide compositions, the mammal is typically administered a dosage level of 0.0001 to 100 mg/kg, more typically 0.001 to 20 mg/kg of its body weight per day. For example, the dose may be 0.3 mg/kg of body weight, 1 mg/kg of body weight, 3 mg/kg of body weight, 5 mg/kg of body weight or 10 mg/kg of body weight or in the range of 1 to 10 mg/kg. An exemplary treatment regimen entails administration 5 times weekly, 4 times weekly, 3 times weekly, 2 times weekly, 1 time every 2 weeks, 1 time every 3 weeks, 1 time every 4 weeks, 1 time monthly, 1 time every 3 months, or 1 time every 6 months. Typically, for intravenous injection or infusion, the dosage may be lower. Multiple dosage regimens can be adjusted to provide the optimum desired response. For example, a single bolus may be administered, multiple separate doses may be administered over time, or the doses may be proportionally reduced or increased depending on the urgency of the treatment situation. It is particularly advantageous to formulate compositions for non-oral administration in dosage unit form for ease of administration and uniformity of dosage. As used herein, the term dosage unit form refers to a plurality of physically discrete units suitable as unitary dosages for administration to a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect with the required pharmaceutical carrier. According to at least some embodiments of the present invention, a specification for the form of the dosage unit is specified and directly depends on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of mixing with such active compounds for the treatment of sensitivity in individuals.
Alternatively, the polypeptide formulation is administered at a level of from 0.0001 to 100 mg/kg of patient weight per day, preferably from 0.001 to 20.0 mg/kg/day, according to any suitable timing regimen. According to at least some embodiments, a therapeutic composition according to at least some embodiments of the present invention may be administered, for example, 3 times a day 1, 2 times a day 1, 1 times a week 3, 2 times a week or 1 time a week, 2 weeks or 3, 4, 5, 6, 7, or 1 time 8 weeks. Also, the composition may be administered in a short time or a long time (e.g., 1 week, 1 month, 1 year, 5 years).
Alternatively, a therapeutic agent such as the compositions disclosed herein may be administered as a sustained release formulation, in which case less frequent administration is required. A dose and a frequency depend on the half-life of the therapeutic agent in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The half-lives of various fusion proteins may vary widely. The dosage and the frequency may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dose is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for their remainder of their lives. In therapeutic applications, it is sometimes desirable to administer a relatively high dose at a relatively short interval until the progression of the disease is reduced or terminated, and preferably until the patient exhibits a partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.
The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may vary in order to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without toxicity to the patient. The dosage level selected will depend upon various pharmacokinetic factors including the activity of the particular composition employed in the present invention, the route of administration, the time of administration, the rate of secretion of the particular compound employed, the duration of treatment, other drugs, the various compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and past medical history of the patient to be treated, and like factors well known in the medical arts.
A "therapeutically effective dose" of a polypeptide as disclosed herein preferably results in a decrease in severity of symptoms of a plurality of diseases, an increase in frequency and duration of disease asymptomatic periods, an increase in longevity, alleviation of disease, or prevention or reduction of damage or disability suffered by the disease.
One of ordinary skill in the art will be able to determine a therapeutically effective amount, particularly in light of the detailed disclosure provided herein, based on a number of factors, such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.
In certain embodiments, the polypeptide, polynucleotide, nucleic acid construct, or cell composition may be administered locally, e.g., by direct injection to a site to be treated. Typically, the injection results in a locally increased concentration of the polypeptide, polynucleotide, nucleic acid construct, or cell composition, which is higher than that achieved by systemic administration. The polypeptide composition may be combined with a matrix as described above to assist in producing a localized increase in the concentration of the polypeptide composition by reducing passive diffusion of the polypeptide out of the site to be treated.
The pharmaceutical compositions of the present invention may be administered with a plurality of medical devices known in the art. For example, in an alternative embodiment, a pharmaceutical composition according to at least some embodiments of the present invention may be administered with a needle hypodermic device, such as those described in U.S. patent publication nos. 5,399,163;5,383,851;5,312,335;5,064,413;4,941,880;4,790,824; or 4,596,556. Examples of well known implants and modules that may be used in the present invention include: U.S. patent publication No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. patent publication No. 4,486,194, which discloses a therapeutic device for transdermal administration of a medicament; U.S. patent publication No. 4,447,233, which discloses a medical infusion pump for delivering medication at a precise infusion rate; U.S. patent publication No. 4,447,224, which discloses a variable flow implantable infusion device for sustained drug delivery; U.S. patent publication No. 4,439,196, which discloses an osmotic drug delivery system having a multi-compartment; and U.S. patent publication No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.
The active compounds can be prepared with a number of carriers for protecting the compounds from too rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. A variety of biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Various methods for preparing such formulations are proprietary or generally known to those skilled in the art. See, e.g., sustained and controlled drug release delivery systems, j.r. robinson, ed., marcel Dekker, inc., new York,1978.
The therapeutic composition may be administered with a plurality of medical devices known in the art. For example, in an alternative embodiment, a therapeutic composition according to at least some embodiments of the present invention may be administered with a needle hypodermic device, such as those described in U.S. patent publication nos. 5,399,163;5,383,851;5,312,335;5,064,413;4,941,880;4,790,824; or 4,596,556. Examples of well known implants and modules that may be used in the present invention include: U.S. patent publication No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. patent publication No. 4,486,194, which discloses a therapeutic device for transdermal administration of a medicament; U.S. patent publication No. 4,447,233, which discloses a medical infusion pump for delivering medication at a precise infusion rate; U.S. patent publication No. 4,447,224, which discloses a variable flow implantable infusion device for sustained drug delivery; U.S. patent publication No. 4,439,196, which discloses an osmotic drug delivery system having a multi-compartment; and U.S. patent publication No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.
In certain embodiments, to determine that the therapeutic compounds according to at least some embodiments of the invention can pass through the Blood Brain Barrier (BBB) (if desired), they can be formulated, for example, in liposomes. Methods for manufacturing liposomes are described, for example, in U.S. patent publications 4,522,811;5,374,548; 5,399,331. The liposomes can comprise one or more moieties that can be selectively delivered to specific cells or organs to enhance delivery of targeted drugs (see, e.g., v.v. ranade (1989) j.clin.pharmacol.29:685). Exemplary targeting moieties include: folic acid or biotin (see, e.g., U.S. patent publication No. 5,416,016 to Low et al); mannosides (Umezawa et al, (1988) biochem. Biophys. Res. Commun. 153:1038); antibody (P.G.Bloeman et al (1995) FEBS Lett.357:140;M.Owais et al (1995) Antimicrob.Agents chemotherS.39:180); a surface active protein A receptor (Briscoe et al (1995) am.J physiol.1233:134); p120 (Schreier et al (1994) J.biol. Chem. 269:9090); see also k.keinanen; M.L.Laukkanen (1994) FEBS Lett.346:123; j. killion; fidler (1994) Immunomethods 4:273.
Formulations for parenteral administration
In a further embodiment, the compositions disclosed herein, including those comprising peptides and polypeptides, are administered by parenteral injection as an aqueous solution. The formulation may also be in the form of a suspension or an emulsion. In general, pharmaceutical compositions are provided that include an effective amount of a peptide or polypeptide, polynucleotide, nucleic acid construct or cell as described herein, and optionally include a pharmaceutically acceptable diluent, preservative, solubilizing agent, emulsifier, adjuvant and/or carrier. Such compositions optionally include one or more of the following: diluents, sterile water, buffered saline of various buffer contents (e.g., tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., polysorbate-20 (TWEEN 20), polysorbate-80 (TWEEN 80), antioxidants (e.g., water-soluble antioxidants such as ascorbic acid, sodium metabisulfite, cysteamine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, oil-soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and metal chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid), and preservatives (e.g., thimerosal, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).
Formulations for topical administration
The various compositions (e.g., polypeptides) disclosed herein may be applied topically. Topical administration does not work well for most peptide formulations, although it may be effective, particularly if applied to the lung, nose, mouth (sublingual, buccal), vagina, or rectal mucosa.
When delivered as an aerosol or as a plurality of spray-dried particles having a aerodynamic diameter of less than about 5 microns, the plurality of compositions may be delivered to the lungs upon inhalation and through the lung epithelial lining to the blood stream.
A wide range of mechanical devices designed for pulmonary delivery of therapeutic products may be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are Ultravent sprayers (marlin crott company, st.louis, mo.); acorn II nebulizer (manufactured by maxster medical science, englewiod, colo.); ventolin scale dose inhaler (ghatti smith, research Triangle Park, n.c.); and Spinhaler powder inhalers (non-liter corporation, bedford, mass.). Nektet, alkmex and Mankea all have inhalable insulin powder formulations approved or in clinical trials, wherein the techniques may be applied in the various formulations described herein.
The various formulations for application to the mucosa are typically spray-dried pharmaceutical particles, which may be incorporated into tablets, gels, capsules, suspensions or emulsions. Standard pharmaceutical excipients are available from any formulator. The oral formulation may be in the form of a chewing gum, gel strip, tablet or lozenge.
Transdermal formulations may also be prepared. These are typically ointments, emulsions, sprays or patches, all of which may be prepared using standard techniques. Transdermal formulations will desirably include penetration enhancers.
Controlled delivery of polymeric matrices
The various compositions (e.g., polypeptides) disclosed herein can also be administered in a controlled release formulation. The controlled release polymer device may be made to systematically release for a long period of time after implantation of a polymer device (rod, cylinder, film, disc) or injection (microparticles). The matrix may be in the form of microparticles, such as microspheres, wherein a plurality of peptides are dispersed in a solid polymer matrix or microcapsule, wherein the core and polymer shell are of different materials, and the peptides are dispersed or suspended in the core, which may be liquid or solid in nature. Microparticles, microspheres, and microcapsules are used interchangeably unless specifically defined herein. Alternatively, the polymer may be formulated as a thin plate or film, ranging from nanometers to 4 centimeters, as a powder manufactured by grinding or other standard techniques, or even as a gel, such as a hydrogel.
Non-biodegradable or biodegradable matrices may be used to deliver the polypeptide or nucleic acid encoding the polypeptide, although biodegradable matrices are preferred. These may be natural or synthetic polymers, but synthetic polymers are preferred because of better characterization of degradation and release profiles. The choice of polymer is based on the time of desired release. In some instances, linear release may be most useful, although in other cases, a pulsed release or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% by weight of water) and is optionally crosslinked with multivalent ions or multiple polymers.
The matrix may be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared by using any method developed for making microspheres for drug delivery, e.g., as described in Mathiowitz and Langer, J.controlled Release,5:13-22 (1987); mathiowitz, et al, reactive Polymers,6:275-283 (1987); and Mathiowitz, et al, J.Appl Polymer ScL,35:755-774 (1988).
The plurality of devices may be formulated for local release for treatment of implanted or injected areas, which typically deliver a dose much smaller than the dose used to treat an entire body, or systemic delivery. These may be implanted or subcutaneously injected into muscle, fat, or swallowed.
If desired, the compositions of some embodiments of the invention may be presented in a package or dispenser device, such as a U.S. Food and Drug Administration (FDA) approved kit, which may comprise one or more unit dosage forms containing the active ingredient. For example, the package may comprise a metal or plastic foil, such as a blister package. The package or dispenser device may be accompanied by an indication of the administration. The package or dispenser may also be provided with a notice associated with the container, the notice being in a form specified by a government agency prescribing the manufacture, use or sale of the pharmaceutical product, the notice reflecting the form of the composition approved by the agency or human or veterinary administration. For example, such notification may be an indication of approved prescription drugs or an approved product information by the U.S. food and drug administration. Compositions comprising a formulation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and identified for use in treating a specified condition, as described in further detail above.
As used herein, the term "about" refers to ± 10%.
The terms "include," comprising, "" include, "" have "and their cognate words are meant to be" including but not limited to.
The term "composition" means "including and limited to".
The term "substantially comprises" means that a composition, method or structure may include additional ingredients, steps and/or portions, provided that the additional ingredients, steps and/or portions do not materially alter the basic and novel characteristics of the composition, method or structure of matter protected by the claims.
As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, and include mixtures thereof.
Throughout this application, various embodiments of this invention can be presented in a range of forms. It should be understood that the description in form of the scope is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual values within that range. For example, a description of a range such as from 1 to 6 should be considered to have a number of subranges specifically disclosed herein, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as a number of individual values in this range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is intended to include any number (fractional or integer) that is referred to as being included within the indicated range. The phrase "ranging between …" is a first indicator number and a second indicator number and "ranging from" to "is a first indicator number" to "a second indicator number is used interchangeably herein and is meant to include the first indicator number and the second indicator number and all decimal and integer numbers therebetween.
The term "method" as used herein refers to means, techniques and procedures for accomplishing a particular task including, but not limited to, those means, techniques and procedures either known or readily developed by practitioners of the chemical, pharmacological, biological, biochemical and medical arts from the known means, techniques and procedures.
When referring to a plurality of specific sequence listings, the references are understood to also encompass a plurality of sequences that substantially correspond to their complementary sequences, as including minor sequence variations due to, for example, sequencing errors, cloning errors, or other changes resulting in base substitutions, base deletions, or base additions, such variations having a frequency of less than 1 out of 50 nucleotides, alternatively less than 1 out of 100 nucleotides, alternatively less than 1 out of 200 nucleotides, alternatively less than 1 out of 500 nucleotides, alternatively less than 1 out of 1000 nucleotides, alternatively less than 1 out of 5000 nucleotides, alternatively less than 1 out of 10,000 nucleotides.
Example
Proof of concept (POC) experiments
Preparation of His-tagged SIRPalpha-41 BBL
For initial proof of concept analysis, a histidine-tagged protein was produced. A cDNA sequence encoding SIRPalpha-41 BBL with 6 His tags was subcloned into a mammalian expression vector. Transfection-level plastid preparation was used to transfect plastids into Expi293 cells or other cell lines. Production of sirpa-41 BBL in the supernatant (on the scale of 100 ml) of the Expi293 expressing cells was assessed by either reducing or non-reducing SDS-PAGE electrophoresis and Western Blot (WB) with an anti-His antibody. Next, his-tagged SIRPalpha-41 BBL was purified from a positive supernatant by affinity-based one step purification (nickel beads). The tagged chimeric proteins were validated by SDS-PAGE electrophoresis and western blot analysis by using multiple specific antibodies against each domain of the molecule (i.e., the extracellular domain of each of sirpa and 41 BBL).
Experiment 1A-production of a His-tagged sirpa-41 BBL fusion protein:
the production of the His-tagged SIRPalpha-41 BBL fusion protein (SEQ ID NO: 5) may be accomplished in an Expi293F cell transfected with a pcDNA3.4 expression vector cloned with sequences encoding the complete fusion protein. The sequence was cloned into the vector using EcoRI and HindIII restriction enzymes, with an increased Kozak (Kozak) sequence at the N-terminus, an artificial signal peptide and 6 His tags, and a stop codon at the C-terminus (SEQ ID NO: 15).
The protein was collected from the supernatant of the cell culture and purified by one step purification through a column under the trade name HisTrapTM FF Crude.
Experiment 1B-the His-tagged sirpa-41 BBL fusion protein made contains two domains:
Material-His tagged SIRPalpha-41 BBL fusion protein (SEQ ID NO: 5) manufactured as described in experiment 1A above, protein tag: spectra BR (Simerfeier technology (Thermo Fisher Scientific), catalog code 26634), anti SIRPalpha (SHPS 1) (Cell Signaling), catalog code 13379), anti 41BB-L (Bayer Wei Sheng (BioVision), 5369-100), mouse anti His monoclonal antibody (gold Style (GenScript), catalog number A00186), secondary goat anti rabbit IgG (H+L) -horseradish peroxidase (HRP) conjugate (1:3333) (R & D, catalog number 170-6515), recombinant hSIRPalpha 0.1 mg/ml (4546-SA-050) R & D, recombinant H41BB-L (TNFSF 9) 0.1 mg/ml (8460 LF) cytosignaling, stripping buffer (Simerfeier technology, catalog code 21059), protein deglycosylation mixture: (NEB p 6044).
Method-proteins (250 nanograms per lane) were treated under denaturing or non-denaturing conditions (boiling at 95 ℃ C. For 5 min in sample buffer containing beta-mercaptoethanol, or without heating in sample buffer without beta-mercaptoethanol, respectively) and separated in a 12% SDS-PAGE electrophoresis gel followed by western blot method. The deglycosylation treatment is achieved by PNGase F enzymes, which are directed by the manufacturer of the protein deglycosylation mixture.
results-Western blot analysis of His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) separated on an SDS-PAGE electrophoresis gel under denaturing conditions followed by immunoblotting with an anti-His-tagged antibody (FIG. 1) or an anti-41 BBL antibody (FIG. 2A) demonstrated the presence of both the N-terminal side of the molecule and the C-terminal side of the molecule. Although the predicted molecular weight of a protein is about 60 kilodaltons based on its amino acid sequence, the protein shifts to a molecular weight of about 85 kilodaltons under denaturing conditions. Such a transition was found to be involved in glycosylation of the protein, as confirmed by treating the protein with PNGase F enzyme, which removes most of the N-linked oligosaccharides from the glycoprotein. After the treatment, a major band of about 60 kilodaltons was observed (fig. 2C).
When separated on an SDS-PAGE electrophoresis under non-denaturing conditions (FIGS. 1 and 2B), the His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) was detected to have the same molecular weight as under denaturing conditions (FIGS. 1, 2A and 2B). Additional higher molecular weight bands were also detected, which were stronger in the non-denaturing condition relative to the denaturing condition. This may suggest the formation of a multimer, possibly a trimer, based on the molecular size of the multimer and the fact that the 41BBL protein naturally tends to form a trimer (Eun-Young et al,2010, J.Biochemical, volume 285, no.12, pp.9202-9210).
Assay of binding of SIRPalpha and 4-1BBL portions of the 1C-chimeras to CD47 and 41BB:
by passing throughTo determine the binding of said sirpa domain of said molecule to CD47 and the binding of said 41BBL domain 41BB of said molecule.
Material-CD 47: FC (Sinogen organism), catalog code 12283-H02H), 41BB: FC (Sinogen organism, catalog code 10041-H03H), his-tagged SIRPal fusion protein (SEQ ID NO: 5) manufactured as described in experiment 1A above; PD1-CD70 protein (SEQ ID NO:6, as a negative control group).
Methods and results-biosensors were preloaded with CD47: fc, which resulted in a stable binding plains (fig. 3A). A rapid binding of His-tagged SIRPalpha-41 BBL to CD47: fc was detected upon subsequent incubation with His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) (FIG. 3A). The same incubation with a control group of proteins PD1-CD70 (consisting of a PD-1 domain fused to CD70, SEQ ID NO: 6) did not result in any binding to CD47: fc (FIG. 3A). Furthermore, when the biosensor was not preloaded with CD47: fc, the His-tagged SIRPalpha-41 BBL was not bound (FIG. 3A, lower line). Once a stable binding plateau is reached, the biosensor is rinsed with culture broth to determine a rate of dissociation of the His-tagged sirpa-41 BBL from CD47: fc. The dissociation of the His-tagged SIRPalpha-41 BBL from the CD47:Fc loaded biosensor was slow, suggesting a stable interaction of SIRPalpha with CD 47.
Binding of the 41BBL unit of His-tagged sirpa-41 BBL was assessed when the biosensor was similarly loaded at 41bb: fc (fig. 3B). As with the sirpa domain, the 41BBL domain of the His-tagged sirpa-41 BBL binds rapidly to its target receptor (fig. 3B), and the dissociation rate of 41BBL/41BB interactions is also very slow, as seen by the limited dissociation that occurs during the final dissociation phase. The treatment control group with PD1-CD70 (SEQ ID NO: 7) did not result in any detectable binding to 41BBL: fc (FIG. 3B), which PD1-CD70 lacks the 41BBL domain. Further, without preloading 41BB: fc, his-tagged SIRPalpha-41 BBL was not detected to bind to the biosensor (FIG. 3B, lower line).
In general, the two domains of the His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) retain functional binding activity to their cognate receptors.
Assay 1D-chimera binding assay of sirpa and 41BBL moiety to CD 47:
binding of the sirpa domain of the molecule to human CD47 was assessed by using HT1080 cells or CHO-K1 cells or another cell line that overexpresses CD47 or a cancer cell line known to exhibit CD47 to a high degree. CD47 depleted cells served as a negative control group. Multiple cells were stained with different concentrations of His-tagged sirpa-41 BBL, followed by staining with a secondary anti-41 BBL antibody. Binding was analyzed by flow cytometry using Fluorescence Activated Cell Sorting (FACS). The use of different concentrations of the chimeras allows the affinity of the molecule to CD47 to be determined. In this binding assay, a recombinant sirpa was also used as a competitor to the sirpa-41 BBL in order to verify the specificity of the binding. A variety of antibodies that block the interaction between sirpa and CD47 may also be used for the same purpose.
Binding of the 41BBL portion of the chimera to human 41BB was tested by using HT1080 cells or another cell line that overexpressed 41 BB. Multiple cells were stained with different concentrations of sirpa-41 BBL, followed by staining with a secondary anti-sirpa antibody, and binding affinity was analyzed by FACs. In this binding assay, a recombinant 41BBL was also used as a competitor to the sirpa-41 BBL in order to verify the specificity of the binding. A variety of antibodies that block interactions between 41BB and 41BBL may also be used for the same purpose.
Material-His tagged SIRPal alpha-41 BBL protein (SEQ ID NO: 5) manufactured as described in experiment 1A above; CHO-WT and CHO-CD47 cell lines (Bommel et al, 2017), fixable active dyes (BD Biosciences, catalogue code 562247), human Fc blockers, true dyes FCX (biological legend, catalogue code 422302), and a variety of antibodies as follows:
method-for the performance assay, cells (0.5M cells/sample) were immunostained with the indicated antibodies, followed by flow cytometry analysis. For binding assays, cells were pre-incubated with human Fc blocker, followed by incubation with different concentrations (0.01 to 50. Mu.g/ml) of the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) on ice for 30 min, followed by immunostaining with antibodies to the "free" arm (41 BBL) of the molecule, immobilization and analysis by flow cytometry.
results-As shown in FIGS. 4A through 4B, CHO-K1-WT did not exhibit either CD47 or 41BB; however, CHO-K1-CD47 cells showed CD47 but not 41BB.
The binding assay showed that His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) bound to CHO-CD47 cells in a dose-dependent manner, however it did not bind to CHO-WT cells (FIGS. 5A-5B).
In general, the N-terminus of the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) may bind to CD47 that is overexpressed on the cell surface.
Experiment 2-41 activation of BB receptor by chimera:
the activation of 41BB receptor by the His-tagged sirpa-41 BBL was tested by using HT1080 cells or another cell line that overexpresses the 41BB receptor. Specifically, the HT1080-41BB cell line over-exhibits 41BB and is known to secrete interleukin-8 (IL-8) upon binding to 41BBL (Wyzgol, et al,2009,The Journal of Immunology). Upon binding of 41BBL to the 41BB receptor on the surface of these cells, a signaling pathway is activated to cause secretion of IL 8. The plurality of cells were incubated in the presence of different concentrations of the His-tagged sirpa-41 BBL, and secretion of IL8 into the medium was determined by ELISA. Oligomerization was tested by adding different concentrations of anti-His tag crosslinking antibodies. As the anti-His tag antibody increases, the chimeric molecule will be crosslinked and form an oligomer to cause an increase in IL8 secretion. Anti-sirpa antibodies can also be used for the same purpose (cross-linking the sirpa portion of the molecule).
The oligomerization can also be tested by co-culturing cells that overexpress the 41BB receptor with HT1080 cells that overexpress human CD47 or with a cancer cell line that highly expresses CD 47. The sirpa-41 BBL binds to CD47 overexpressed on HT1080 cells or on cancer cells, and the 41BBL moiety is presented to HT1080 cells overexpressing 41BB receptors. Because of this behavior in the vicinity of multiple molecules, the need for oligomerization can be fulfilled.
The 41BBL receptor can be compared by activation of His-tagged sirpa-41 BBL with a portion thereof, i.e., activation of recombinant sirpa or 41BBL, alone or in combination.
Material-His tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5), HT1080-41BB cell line (Lang et al 2015), IL-8ELISA kit (catalog code D8000C, R & D), DMEM (catalog code 01-055-1A, biological industry (Biological industries)), fetal Bovine Serum (FBS) (catalog codes 10270106, lei Lien (Rhenium)), AIM V (serum free Medium) (Simer Feier technology) manufactured as described in experiment 1A above.
method-HT 1080-41BB cells (5000 per well) were incubated with different concentrations of His-tagged SIRPal-41 BBL protein (SEQ ID NO: 5) for 24 hours. The concentration of IL-8 in the supernatant was determined by the IL-8ELISA kit, which was according to the manufacturer's method steps. Serum-free medium was used in some experiments to eliminate the relatively high background values detected using medium with FBS.
Results-multiple independent experiments showed the functionality of sirpa-41 BBL: his-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) triggers Tumor Necrosis Factor Receptor (TNFR) signaling in FBS-containing medium (FIG. 6) and in serum-free medium (FIG. 7) in a dose-dependent manner as determined by IL8 secreted by HT1080-41BBL cells.
Experimental 3-T cell activation by sirpa-41 BBL:
the effect of sirpa-41 BBL on T cell activation was tested by using T cells in human healthy donor PBMCs or by using human TILs. The T cells were first co-cultured with human cancer cells and treated with anti-CD 3 and anti-Epcam 1 bi-specific antibodies to induce T cell activation, followed by sirpa-41 BBL treatment. The anti-CD 3/Epcam1 antibody carries a first signal for activating T cells directed against cancer cells exhibiting Epcam 1. The sirpa-41 BBL molecule interacts with CD47 expressed on the surface of cancer cells, which promotes expression and oligomerization of the molecule, thereby allowing the 41BBL moiety to interact with 41BB receptors on T cells, and to deliver a second co-stimulatory signal to the T cells. The degree of activation of the T cells is determined by measuring a plurality of parameters; first, the performance of a plurality of activation markers (e.g., CD25, CD69, CD62L, CD137, CD107a, PD1, etc.) on the surface of the T cells is tested. The performance of activation markers was tested by staining cells with a number of specific antibodies and flow cytometry (FACS). A second method for determining T cell activation is by measuring secretion of inflammatory cytokines (e.g., IL2, IL6, IL8, INFγ, etc.). Secretion of inflammatory cytokines was tested by ELISA. Proliferation of T cells was measured by pre-staining T cells with carboxyfluorescein succinimidyl ester (CFSE) and determining cell bias by dilution of CFSE measured by FACS. Another parameter to be tested is killing of the cancer cells by pre-labelling the cancer cells with a Calcine-AM reagent and measuring the release of calcein into the medium by using a luminescent plate reader.
The effect of sirpa-41 BBL on TILs activation was tested on TILs extracted from tumors, followed by co-culture with the tumor cancer cells and treatment with sirpa-41 BBL. The first signal to activate T cells passes through MHC class i: the peptide-TCR (T cell receptor) pathway is carried by the cancer cell. The sirpa-41 BBL fusion protein interacts with CD47 expressed on the tumor cells, which promotes expression and oligomerization of the molecule, and thus allows the 41BBL moiety to interact with 41BB receptor on T cells and deliver a second co-stimulatory signal to the T cells. The extent of activation of the TILs and killing of tumor cells was determined in the same manner as described (activation markers, cytokine secretion, proliferation and killing of tumor cells).
Activation of T cells by His-sirpa-41 BBL can be compared to activation of a portion thereof, i.e., recombinant sirpa or 41BBL, alone or in combination.
Experiment 3A-SIRPalpha-41 BBL protein demonstrates the costimulatory activity of T cells:
Material-His tagged SIRPal alpha-41 BBL protein (SEQ ID NO: 5) manufactured as described in experiment 1A above; HT1080-41BB, CHO-WT, CHO-K1-CD47 and DLD cell lines (Bommel et al 2017,Lang et al 2015,ATCC-CCL-221), freshly isolated human T cells, IL8Elisa kit (R & D, catalog code DY 208), CD47: FC (Sainoorganism, catalog code 12283-H02H), anti-CD 3/anti-CD 28 activating beads (Lefu life technologies (Life Technologies), catalog code 11131D), anti-CD 25 antibodies (immunological Tools, catalog code 21270256), lymphocyte isolation (Lymphoprep) (Stem cell technologies (Stemcell), 07851), names: CD14 microbeads, human (Miltenyi Biotec, 130-050-201).
Methods and results when single cultured 41BB transduced HT1080 cells (HT 1080-41 BB) were treated with His tagged SIRPalpha-41 BBL (SEQ ID NO: 5), a minimal production of IL8 was detected after 24 hours of incubation (FIG. 8A). Likewise, treatment with His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) minimally induced secretion of IL8 when HT1080-41BB cells were mixed with wild type CHO cells (FIG. 8A). However, treatment of mixed culture HT1080-41BB with CHO-CD47 (such that the SIRPalpha domain binds to CHO-CD47 and exhibits cross-linking of 41BBL with HT1080-41 BB) with His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) triggered a strong increase in IL-8 secretion, which peaked at 2000 picograms/milliliter (FIG. 8B). Thus, binding of the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) to CD47 is beneficial in order to stimulate secretion of IL-8 upon 41BBL/41BB interaction.
Next, the potential of the 41BBL domain through His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) was assessed for induction of T cell activation. PBMCs are isolated from blood of healthy donors by Lymphoprep, which is according to the manufacturer's instructions. Next, a plurality of cells were stained with CD14 microbeads and T cells were isolated with MACS sorting system according to the manufacturer's instructions. To this end, freshly isolated T cells were added to CD47-Fc coated discs and activated with sub-optimal concentrations of anti-CD 3/anti-CD 28 activation beads for 3 days. After treatment, a significantly increased proportion of activated cd25+ T cells was detected in cells treated with the His-tagged sirpa-41 BBL (fig. 8C), which had an optimal induction at about 2.5 μg/ml. In subsequent mixed culture of DLD-1 and T cells, treatment with His-tagged SIRPal-41 BBL protein (SEQ ID NO: 5) increased the proportion of CD25+ T cells (FIGS. 8C-8E), indicating that SIRPal-41 BBL protein may activate T cells. Thus, binding of the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) to CD47 enables 41BBL/41BB mediated co-stimulation and T cell activation to occur.
Overall, these data provide clear evidence that the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) acquires 41 BBL-mediated co-stimulatory activity that enhances T cell activation after CD 47-mediated binding.
Experiment 3B-SIRPalpha-41 BBL protein enhances activation of human PBMCs:
Material-His tagged SIRPal alpha-41 BBL protein (SEQ ID NO: 5) manufactured as described in experiment 1A above; INF-gamma ELISA kit [ catalog code 900-TM27, catalog code 900-T00-Elisa buffer kit (TMB) ], RPMI (catalog code 01-100-1A, bio-industry), FBS (catalog code 12657-029, gibco)), L-glutamine (catalog code 25030-24, gibco), pen/Strep (catalog code 15140-122, gibco), leaf purified anti-human CD3 (catalog code BLG-317315, bio-legend), recombinant human IL2 (catalog code 202-IL-500, R & D system), human peripheral mononuclear blood cells (PBMCs), isolated from peripheral blood of healthy donors by Ficoll-Paque (catalog code 17-1440-03, GE healthcare (GE healthcare)), anti-CD 47 antibodies (catalog code MCA2514A647, bule), anti-41L antibodies (human catalog 311504, bio-legend), 4-11 leukemia cells (ATCC-201m, abra 7).
Method-expression of CD14 by MV4-11 cells and binding to His-tagged sirpa-41 BBL was tested by flow cytometry. Human PBMCs were isolated from peripheral blood of healthy donors by using Ficoll-Paque (Grienvic et al 2016). Next, PBMCs were incubated with different concentrations of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) in the presence of anti-CD 3 (30 picograms/ml) or anti-CD 3 plus IL2 (1000 units/ml) for 40 hours. The experiment was performed with or without co-cultivation with human cancer cell line MV4-11, which exhibits CD47 (ratio 1:1). The concentration of INF-gamma in the cell supernatant was determined by INF-gamma ELISA kit, which was according to the manufacturer's procedure.
Results-human PBMCs, including natural killer cells, natural killer T cells, CD4+ and CD8+ acting Th1 cells, are known to secrete pro-inflammatory interferon-gamma (INF-gamma) in response to activation. Activation of a T cell requires two signals: the attachment of T Cell Receptors (TCRs) to Major Histocompatibility Complex (MHC)/peptide complexes on Antigen Presenting Cells (APCs), and the crosslinking of co-stimulatory receptors on T cells to corresponding ligands on APCs. 41BB is a T cell costimulatory receptor induced by the ligation of 41 BBL. 41BB transmits an effective co-stimulatory signal to CD8+ and CD4+ T cells to promote their expansion, survival, differentiation, and expression of cytokines. Its ligand, 41BB,41BBL, is a membrane protein that provides a costimulatory signal to T cells.
In this experiment, the functionality of sirpa-41 BBL molecules in enhanced activated human PBMCs was assessed.
CD47 was present on the expression of MV4-11 cells, and His-tagged SIRPal-41 BBL protein (SEQ ID NO: 5) was bound to these cells in a dose-dependent manner (FIGS. 9A-9B). Incubation of MV4-11 cells with different concentrations of His-tagged SIRPal-41 BBL protein (SEQ ID NO: 5) for up to 72 hours did not show any direct killing effect (FIG. 9C).
The addition of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) in a dose-dependent manner enhanced activation of the PBMCs, as can be seen by the increase in INF-gamma secretion from the PBMCs by stimulation with anti-CD 3 antibodies with or without IL2 addition (FIG. 10A).
Co-culture of PBMCs with human cell lines MV4-11 results in secretion of INF-gamma by stimulation of the cells with anti-CD 3 antibodies, probably due to direct stimulation of the PBMCs cells by the MV4-11 cells. Treatment with the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) had a modest effect that was more pronounced when added with IL 2.
In general, his-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) enhances activation of human PBMCs, as seen by secretion of IFN-gamma.
Experiment 4-SIRP alpha-41 BBL effect on pellet and macrophage:
sirpa is an inhibitory receptor that is displayed on the cell surface of, for example, phagocytes, and the ligand CD47 of sirpa is widely expressed on a variety of tumor cells. Once sirpa is engaged by CD47, sirpa transmits a "do not eat me" signal to phagocytes. Through the interaction of sirpa-41 BBL with CD47, it should block the endogenous interaction of CD47 with sirpa, and by blocking the "do not eat me" signal will allow phagocytes to engulf the tumor cells.
The effect of sirpa-41 BBL on pellet, macrophages and other phagocytes was tested in vitro in a co-culture assay by co-culturing pellet or M1 macrophages from healthy donors with fluorescent labeled cancer cells exhibiting CD 47. Sirpa-41 BBL was added to the co-culture at different concentrations and phagocytosis was determined by measuring the amount of fluorescence taken up by the pellet or M1 macrophages using flow cytometry. The phagocytes are identified and the cancer cells are distinguished by a plurality of specific surface markers, such as CD11 b.
In this experiment, the phagocytosis may be enhanced by the use of various therapeutic anti-tumor antibodies (e.g., rituximab, cetuximab, trastuzumab, alemtuzumab, etc.).
Similar experiments can also be performed by co-culturing autologous pellet from cancer patient with primary autologous malignant cells.
Material-His tagged SIRPal alpha-41 BBL protein (SEQ ID NO: 5) manufactured as described in experiment 1A above, human leukocytes isolated from peripheral blood, tumor cell lines: human B-cell lymphoma cells (BJAB), human diffuse large B-cell lymphoma cells (U2932), human Burkitt's lymphoma cells (Raji), human B-cell lymphoma (Sudhl 6), human colorectal adenocarcinoma epithelial cells (DLD 1), human lung cancer cells (H292), human pharyngeal squamous cell carcinoma cells (FADU), human ovarian cancer cells (OVCR), human acute bone-associated leukemia cells (MOLM 13), human chronic bone-associated leukemia cells (K562), human acute bone-associated leukemia cells (ocial 3), human acute pre-myelogenous leukemia cells (HL 60) (ATCC), vybrant di (invitrogen life technologies, catalog code V22887), cell division stain V450 (sampler fei, 65-0842-85), lymphrep (stem cell technologies, 07851), recombinant human sirpα (sirnoorganism, catalog code 11612-H08H), recombinant human 41BBL (R & D system, 224L/CF).
Method and results-white blood cells are separated from the peripheral blood of healthy donors. Briefly, whole blood was mixed with Phosphate Buffered Saline (PBS) containing EDTA at a ratio of 1:1 and with lymphoprep. To obtain neutrophils, PBMCs were removed after lymphoprep and cell pellet was harvested. The cell pellet was mixed with red blood cell lysate at a ratio of 1:10 and incubated at 4 ℃ for 30 to 45 minutes. Then, neutrophils were harvested by centrifugation at 450 g/5 min and washed twice with PBS containing EDTA. The isolated leukocytes were mixed with tumor cells, which had been pre-stained with vybrant DiD, at a ratio of 1:1. Flow cytometry was then used to assess the uptake of the tumor cells by pellet spheres (see fig. 11A for gating strategies). In such mixed culture, the pellet minimizes phagocytic tumor cells in the absence of any stimulus (fig. 11A). However, when leukocytes were co-cultured with Ramos B-NHL cells to increase the concentration of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5), a significant dose-dependent phagocytosis induced by pellet was detected, which achieved an optimal effect at 2.5 μg/ml (FIGS. 11A-11B). For a group of B cell lymphoma cell lines, single agent treatment with His-tagged SIRPal-41 BBL protein (SEQ ID NO: 5) detected similar pro-phagocytic activity, yielding 15% to 20% increase in phagocytosis compared to untreated cultures (FIG. 11C).
The mixed culture was then treated with a combination of His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) and rituximab, an anti-CD 20 antibody. As shown in fig. 11D, treatment with rituximab at very low suboptimal concentrations has triggered phagocytosis by pellet balls, the extent of which depends on the donor and cell strain of the white blood cells (fig. 11D, white diamond). However, rituximab binding treatment with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) further increased phagocytic uptake of all B-NHL cell lines to be tested (FIG. 11D, black diamond).
To determine if sirpa-41 BBL binding to other therapeutic antibodies would similarly enhance phagocytosis, a panel of cancer cell lines was mixed with the white blood cells. Phagocytosis by pellet was assessed after treatment with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) with or without the anti-Epidermal Growth Factor Receptor (EGFR) antibody cetuximab. In all cancer cell lines to be tested, single agent treatment with His-tagged SIRPal-41 BBL protein (SEQ ID NO: 5) has strongly triggered phagocytic uptake by cancer cells (FIG. 11E), with pellet spheres phagocytosis to DLD cells up to 80%. Following His-tagged sirpa-41 BBL binding treatment with cetuximab, phagocytic uptake of cancer cells was increased, even further (fig. 11F): for example, greater than 90% are phagocytosed in squamous cell carcinoma line FaDu, suggesting an additive or even synergistic effect of cetuximab with sirpa-41 BBL on this or other cell lines.
Subsequent analysis of phagocytosis of some acute myelogenous leukemia cell lines revealed that most of these cell lines were unresponsive to treatment with His-tagged SIRPalpha-41 BBL protein alone (SEQ ID NO: 5) (FIG. 11G). An notable exception here is an acute promyelocytic leukemia cell line HL60, whose phagocytosis increases by about 10 to 20% (depending on the donor) after 2 hours of incubation compared to untreated cultures. Particle ball-mediated phagocytosis of K562 (chronic bone-associated leukaemia) and Oci-AML3 (acute bone-associated leukaemia) was also strongly increased after prolonged treatment for up to 24 hours compared to untreated cultures, however, at this prolonged time point, HL60 uptake was already very high in untreated control cultures (figure 11H). Increasing the dose of His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) at the 2 hour time point further increased phagocytosis of HL60, while MOLM13 phagocytosis did not (FIG. 11I).
Importantly, primary AML blast cells obtained from AML patients with complex karyotypes were phagocytized after treatment with His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) in a dose-dependent manner (FIG. 11J). In the extended group of allogeneic pellet spheres from five healthy donors, these primary AML blast cells were strongly phagocytosed within 2 hours compared to untreated cultures, with an increase in median phagocytosis of about 35% (fig. 11K). Furthermore, after 24 hours, the phagocytosis was still enhanced after treatment with His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5), although the phagocytosis was increased in untreated cultures (FIG. 11K).
To further demonstrate the potential of sirpa-41 BBL for stimulating phagocytosis, a similar experiment was performed by using macrophages derived from mononuclear spheres. Briefly, PBMCs enriched with multiple mononuclear spheres were isolated from healthy donors by MACS sorting using CD14 magnetic microbeads (the spurious Tenivey technology). The plurality of mononuclear spheres was placed in RPMI 1640 medium + 10% fetal bovine serum (FCS) supplemented with granular-sphere macrophage colony-stimulating factor (GM-CSF) (50 nanograms/ml) and macrophage colony-stimulating factor (M-CSF) (50 nanograms/ml) for 7 days to differentiate into macrophages (M0). To produce type I macrophages, M0 cells were primed with lipopolysaccharide and IFN-gamma for an additional 24 hours. Next, macrophages differentiated in vitro were mixed with B-cell lymphoma cell line U2932, which U2932 was pre-stained with cell proliferation stain V450. Macrophages were mixed with U2932 for 2 hours; and phagocytic uptake of cancer cells was measured by fluorescence microscopy (a plurality of representative micrographs are shown in fig. 12A, with dark arrows indicating living cancer cells and white arrows indicating cancer cells phagocytosed in macrophages). Treatment with His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) had a limited activity in this setting (FIG. 12B). Treatment with rituximab induced a strong increase in phagocytosis (fig. 12C, white diamond), which was further enhanced in most donors by co-treatment with His-tagged sirpa-41 BBL (SEQ ID NO: 5) with various concentrations of rituximab (fig. 12C, black diamond).
Further, in experiments with a mixed culture of primary B-cell chronic lymphocytic leukemia (B-CLL) and macrophages autologous from the patient, treatment with the anti-CD 52 antibody alemtuzumab alone (these blast cells were highly positive for CD 54) induced nearly 40% of macrophage-induced phagocytosis (fig. 11D); treatment with His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) alone has a minimal level of pro-phagocytic activity; however, in these primary samples, binding of His-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) to alemtuzumab enhanced phagocytosis (about 20% enhancement compared to alemtuzumab treatment) (FIG. 12D).
And, the His-tagged SIRPalpha-41 BBL fusion protein (SEQ ID NO: 5) has an effect on phagocytes that is superior to that of soluble SIRPalpha alone, soluble 41BBL alone or a combination thereof (FIG. 11L).
In general, his-tagged SIRPalpha-41 BBL (SEQ ID NO: 5) enhances pellet-and macrophage-mediated phagocytic uptake by various malignant cell types, including primary malignant cells, through its SIRPalpha domain, particularly in combination with various clinically used therapeutic monoclonal antibodies for treatment.
Experiment 5-in vivo concept verification:
sirpa-41 BBL was tested in vivo in the targeting and activation of T cells, natural killer cells and B cells, and in the mouse model of the activation of phagocytes and dendritic cells. The mouse His-SIRPalpha-41 BBL fusion protein is produced and purified in the same manner as the tag protein of the human molecule. The mouse tumor pattern is generated by injecting mouse cancer cells into the mouse, which are known to form tumors that express mouse CD 47. Mice were treated with a His-tagged sirpa-41 BBL fusion protein from mice or a His-tagged sirpa-41 BBL fusion molecule from humans. Tumor size, survival of mice, and inflammatory response at the tumor site can be monitored.
Similar experiments can be performed in humanized mouse mode by using human tumors. This pattern was constructed by mice lacking any mouse immune system (Nude/SCID/NSG mice). These mice establish a class of human immune systems by injection of human T cells alone, PBMCs, or by using genetically engineered mice possessing a fully humanized immune system. The mice were vaccinated with human cancer cells and treated with human His-tagged sirpa-41 BBL molecules. Tumor size, survival of mice, and inflammatory response at the tumor site in this mode can also be monitored. The in vivo efficacy of the His-tagged sirpa-41 BBL can be compared to the efficacy of a portion thereof, i.e., recombinant sirpa or 41BBL, alone or in combination.
Experiment 5A-SIRPalpha-41 BBL protein inhibits tumor growth in mice vaccinated with syngeneic colon cancer:
material-autoclaved mouse diet and litter (Shniff, sorst, germany), female Balb/C mice (Janvier, saint Bethleten, france), CT-26 mouse colon cancer cell beads (ATCC-CRL-2638), anti-mouse PD-1 antibodies (BioXcell, centipeda, USA), his-tagged SIRPal alpha-41 BBL protein (SEQ ID NO: 5) manufactured as described in experiment 1A above, PBS.
Method-mice were individually maintained in ventilated cages in groups of 4 mice per cage. The mice were free to receive autoclaved food with litter and acidified (ph 4.0) tap water. The animal farm was assembled for an automatic 12 hour light/dark adjustment, temperature adjustment at 22.+ -. 2 ℃ and relative humidity of 50.+ -. 10%. Female Balb/C mice were inoculated subcutaneously 1X 10 6 CT-26 cells were counted and treatment was started after 3 days. Following random assignment, 10 animals of each group were dosed intravenously with His-tagged SIRPal alpha-41 BBL protein (SEQ ID NO: 5) (100. Mu.g/injection) or its soluble buffer (PBS) 2 times per week 3 times. 5 mg/kg of anti-mouse PD1 was used as a treatment control group at the same time course (FIG. 13A). All administrations were carried out in the morning without anesthesia. Tumor volumes were determined 3 times per week by using caliper measurements, and individual volumes were calculated by the formula: volume= ([ width ] ]2x length)/2. All animal experiments were performed in accordance with the rules of the british institutional review of Cancer for animal welfare (Workman et al, national institutional review of Cancer committee. Welfare guidelines for animals in Cancer research: br J Cancer 2010;102: 1555-77) and German animal protection lawsAnd approved by the local authorities (Gen 0030/15).
Results-in this experiment, the effects of sirpa-41 BBL in vivo were assessed by using the CT-26 mouse colon cancer model. Treatment of CT-26 vaccinated mice with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) significantly reduced tumor volume (up to about 36%) (FIG. 13B).
Experiment 5B-SIRP alpha-41 BBL protein can effectively treat mice inoculated with isogenic leukemia tumors:
materials-autoclaved mouse diet and litter (Shniff, sorst, germany), female DBA/2 mice (Janvier, st. Beten, france), P388 leukemia cell line (Max-Debybuk-molecular medicine center, berlin, germany), anti-mouse PD1 antibodies (BioXcell, celebur, USA), his-tagged SIRP alpha-41 BBL protein (SEQ ID NO: 5), PBS.
Method-mice were individually maintained in ventilated cages in groups of 4 mice per cage. Freely receiving autoclaved food with litter and acidified (ph 4.0) tap water. The animal farm was assembled for an automatic 12 hour light/dark adjustment, temperature adjustment at 22.+ -. 2 ℃ and relative humidity of 50.+ -. 10%. Female DBA/2 mice were inoculated intraperitoneally 1X 10 6 P338 cells and treatment was started after 1 day. Following random assignment, 10 animals of each group were dosed intravenously with His-tagged SIRPal-41 BBL protein (SEQ ID NO: 5) (100. Mu.g/injection) or its soluble buffer (PBS) 4 times every 2 days 1. 5 mg/kg of anti-mouse PD1 was used as a treatment control group at the same time course (FIG. 13A). All administrations were carried out in the morning without anesthesia. Mice suffering from P388 were weighed daily and sacrificed once they became moribund; and the volume of abdominal water was measured. Furthermore, spleen and liver of each mouse were removed and weighed. All animal experiments were performed in accordance with the regulations of the british institutes of Cancer research on animal welfare (Workman et al, national institutes of Cancer, guidelines for welfare of animals in Cancer research, br J Cancer 2010; 102:1555-77) and german animal protection laws and were approved by the local authorities (Gen 0030/1).
Results-in this experiment, the effects of sirpa-41 BBL in vivo were assessed by using the P338 ascites mouse leukemia model. In this mode, spleen weight is a marker of disease severity due to the fact that spleen acts as a draining lymph node for ascites. Treatment of mice vaccinated with P338 mice leukemia with the His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) was effective, as seen by the preferred prognosis of the disease indicated by the significant decrease in spleen weight (19%, P value=0.03) (FIG. 14B).
Experiment 5C-sirpa-41 BBL protein reduced tumor burden in bone following (BM) of NSG mice vaccinated with human leukemia tumors:
material-female NSG mice (NOD-scid gamma mice) 12 to 16 weeks old, MV4.11 acute bone-associated leukemia cell line (ATCC-CRL-9591), anti-mouse CD45 antibody (e bioscience, san Diego, USA), anti-human CD123 antibody (BD Pharmingen, USA), his-tagged SIRP alpha-41 BBL protein (SEQ ID NO: 5) manufactured as described in experiment 1A above, PBS. Human PBMCs were isolated from peripheral blood of healthy donors by using the Ficoll-Paque method (Sigma-Aldrich Israel).
Method-mice were individually maintained in ventilated cages in groups of 5 mice per cage. The mice were free to receive autoclaved food with litter and acidified (ph 4.0) tap water. The animal farm was assembled for an automatic 12 hour light/dark adjustment, temperature adjustment at 22.+ -. 2 ℃ and relative humidity of 50.+ -. 10%. Female NSG mice were irradiated with 200 rad (rad) for 24 hours and then exposed to a radiation containing 7.8x10 6 Is injected intravenously into the veins of the tail with PBS having a total volume of 200 microliters. Mice were vaccinated 1.5x10 by tail vein after 13 days 6 Human PBMCs and treatment was initiated after 4 hours. Following randomized assignment, 5 animals of each group were dosed (on days 13, 15, 17 and 19) 4 times every 1 day (EOD) with His-tagged SIRPal-41 BBL protein (SEQ ID NO:5, 100 μg/injection) or its soluble buffer (PBS) for intraperitoneal injection (FIG. 15A). All ofThe administration of (c) is carried out in the morning without anesthesia. Mice were sacrificed 24 hours after the last injection and blood, bone in-line (BM) and spleen were collected. Spleen was weighed and extracted bone following cells were analyzed by FACS after staining with hCD45, mCD45 and CD 123.
All animal experiments were conducted in accordance with the regulations of the Hadassa (Hadassa) medical center committee on animal welfare and israel animal protection laws and approved by the local main authorities.
Results-treatment of NSG mice vaccinated with MV4.11 and irradiated, which had not been reconstituted with human PBMCs, with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) did not affect the number of leukemia cells in bone following (FIG. 15B). Treatment of NSG mice vaccinated with MV4.11 with human PBMCs reduced tumor burden (10% reduction) in bone following (fig. 15B), and did not increase spleen weight (fig. 15C). However, treatment of NSG mice vaccinated with MV4.11 with His-tagged SIRPalpha-41 BBL protein (SEQ ID NO: 5) along with human PBMCs reduced tumor burden in bone (38%) and also increased spleen weight (FIG. 15C). The increase in spleen seen only when His-tagged sirpa-41 BBL protein was bound to human PBMCs indicated an increase in Graft Versus Leukemia (GVL) effect in bone following, which was also associated with a decrease in the number of leukemia cells in bone following (fig. 15B-15C), p-value = 0.01.
Sequence listing
<110> Karl medical Co., ltd
Sha Ni Norhm
Gotz blue, about West
Jenesyl Eihharlequin, mi Haer
Bladed, ehde temperature
Kaminsky, yidu
<120> SIRPa-41 BBL fusion proteins and methods of use thereof
<130> 72308
<150> US 62/442,469
<151> 2017-01-05
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 549
<212> PRT
<213> Artificial sequence
<220>
<221> PEPTIDE
<222> (1)..(549)
<223> amino acid sequence of chimeric protein (SIRPalpha-G-41 BBL)
<400> 1
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala
115 120 125
Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly
130 135 140
Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
145 150 155 160
Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
165 170 175
Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val
180 185 190
His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
195 200 205
Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro
210 215 220
Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn
225 230 235 240
Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
245 250 255
Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val
260 265 270
Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
275 280 285
Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu
290 295 300
His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
305 310 315 320
Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly
325 330 335
Ser Asn Glu Arg Asn Ile Tyr Gly Ala Cys Pro Trp Ala Val Ser Gly
340 345 350
Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly
355 360 365
Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln
370 375 380
Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly
385 390 395 400
Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr
405 410 415
Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys
420 425 430
Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val
435 440 445
Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro
450 455 460
Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu
465 470 475 480
Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly
485 490 495
Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His
500 505 510
Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr
515 520 525
Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro
530 535 540
Ser Pro Arg Ser Glu
545
<210> 2
<211> 343
<212> PRT
<213> homo sapiens
<400> 2
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala
115 120 125
Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly
130 135 140
Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
145 150 155 160
Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
165 170 175
Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val
180 185 190
His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
195 200 205
Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro
210 215 220
Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn
225 230 235 240
Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
245 250 255
Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val
260 265 270
Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
275 280 285
Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu
290 295 300
His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
305 310 315 320
Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly
325 330 335
Ser Asn Glu Arg Asn Ile Tyr
340
<210> 3
<211> 205
<212> PRT
<213> homo sapiens
<400> 3
Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala
1 5 10 15
Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro
20 25 30
Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala
35 40 45
Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro
50 55 60
Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp
65 70 75 80
Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe
85 90 95
Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val
100 105 110
Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala
115 120 125
Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg
130 135 140
Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly
145 150 155 160
Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala
165 170 175
Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr
180 185 190
Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
195 200 205
<210> 4
<211> 548
<212> PRT
<213> Artificial sequence
<220>
<221> PEPTIDE
<222> (1)..(548)
<223> amino acid sequence of chimeric protein (SIRPalpha-41 BBL, no connecting chain)
<400> 4
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala
115 120 125
Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly
130 135 140
Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
145 150 155 160
Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
165 170 175
Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val
180 185 190
His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
195 200 205
Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro
210 215 220
Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn
225 230 235 240
Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
245 250 255
Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val
260 265 270
Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
275 280 285
Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu
290 295 300
His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
305 310 315 320
Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly
325 330 335
Ser Asn Glu Arg Asn Ile Tyr Ala Cys Pro Trp Ala Val Ser Gly Ala
340 345 350
Arg Ala Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro
355 360 365
Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly
370 375 380
Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro
385 390 395 400
Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly
405 410 415
Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala
420 425 430
Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala
435 440 445
Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu
450 455 460
Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro
465 470 475 480
Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg
485 490 495
Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr
500 505 510
Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val
515 520 525
Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser
530 535 540
Pro Arg Ser Glu
545
<210> 5
<211> 554
<212> PRT
<213> Artificial sequence
<220>
<221> PEPTIDE
<222> (1)..(554)
<223> amino acid sequence of His-tagged SIRPalpha-41 BBL
<400> 5
His His His His His His Glu Glu Glu Leu Gln Val Ile Gln Pro Asp
1 5 10 15
Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr
20 25 30
Ala Thr Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala
35 40 45
Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro
50 55 60
Arg Val Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe
65 70 75 80
Ser Ile Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr
85 90 95
Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser
100 105 110
Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val
115 120 125
Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe
130 135 140
Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp
145 150 155 160
Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro
165 170 175
Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val
180 185 190
Leu Thr Arg Glu Asp Val His Ser Gln Val Ile Cys Glu Val Ala His
195 200 205
Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu
210 215 220
Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg
225 230 235 240
Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro
245 250 255
Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr
260 265 270
Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp
275 280 285
Met Ser Trp Leu Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys
290 295 300
Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser
305 310 315 320
His Asp Leu Lys Val Ser Ala His Pro Lys Glu Gln Gly Ser Asn Thr
325 330 335
Ala Ala Glu Asn Thr Gly Ser Asn Glu Arg Asn Ile Tyr Gly Ala Cys
340 345 350
Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ser Pro
355 360 365
Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu
370 375 380
Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val
385 390 395 400
Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala
405 410 415
Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu
420 425 430
Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu
435 440 445
Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala
450 455 460
Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala
465 470 475 480
Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala
485 490 495
Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu
500 505 510
Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu
515 520 525
Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile
530 535 540
Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
545 550
<210> 6
<211> 312
<212> PRT
<213> Artificial sequence
<220>
<221> PEPTIDE
<222> (1)..(312)
<223> amino acid sequence of PD1-CD70
<400> 6
His His His His His His Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg
1 5 10 15
Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu
20 25 30
Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser
35 40 45
Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys
50 55 60
Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg
65 70 75 80
Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val
85 90 95
Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile
100 105 110
Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu
115 120 125
Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro
130 135 140
Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Gly Gln Arg Phe
145 150 155 160
Ala Gln Ala Gln Gln Gln Leu Pro Leu Glu Ser Leu Gly Trp Asp Val
165 170 175
Ala Glu Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu
180 185 190
Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro
195 200 205
Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met
210 215 220
Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser
225 230 235 240
Arg His His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser
245 250 255
Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile
260 265 270
Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr
275 280 285
Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe
290 295 300
Phe Gly Val Gln Trp Val Arg Pro
305 310
<210> 7
<211> 1665
<212> DNA
<213> Artificial sequence
<220>
<221> gene
<222> (1)..(1665)
<223> nucleic acid sequence of SIRPalpha-41 BBL (with His tag)
<400> 7
caccatcatc accaccatga agaggaactg caagtgatcc agcctgacaa gagcgtgctg 60
gtggctgctg gcgaaacagc cacactgaga tgtaccgcca cctctctgat ccctgtgggc 120
cctatccagt ggtttagagg cgctggacct ggcagagagc tgatctacaa ccagaaagag 180
ggacacttcc ccagagtgac caccgtgtcc gacctgacca agcggaacaa catggacttc 240
agcatccgga tcggcaacat cacccctgcc gatgccggca cctactactg cgtgaagttc 300
agaaagggca gccccgacga cgtcgagttt aaaagcggag ccggcacaga gctgagcgtg 360
cgggctaaac cttctgctcc tgtggtgtct ggacctgccg ctagagctac acctcagcac 420
accgtgtctt ttacctgcga gagccacggc ttcagcccca gagatatcac cctgaagtgg 480
ttcaagaacg gcaacgagct gtccgacttc cagaccaacg tggaccctgt gggagagagc 540
gtgtcctaca gcatccacag cacagccaag gtggtgctga cccgggaaga tgtgcactcc 600
caagtgattt gcgaggtggc ccacgttacc ctgcaaggcg atcctctgag aggcaccgcc 660
aatctgagcg agacaatccg ggtgccacct acactggaag tgacccagca gcctgtgcgg 720
gccgagaatc aagtgaacgt gacctgccaa gtgcggaagt tctaccctca gagactgcag 780
ctgacctggc tggaaaacgg caatgtgtcc agaaccgaga cagccagcac cgtgaccgag 840
aacaaggatg gcacctacaa ttggatgagc tggctgctcg tgaatgtgtc tgcccaccgg 900
gacgatgtga agctgacatg ccaggtggaa cacgatggcc agcctgccgt gtctaagagc 960
cacgacctga aggtgtccgc tcatcccaaa gagcagggct ctaatactgc cgccgagaac 1020
accggcagca acgagagaaa tatctacggc gcttgtcctt gggccgtttc tggcgctaga 1080
gcctctcctg gatctgccgc ttctcccaga ctgagagagg gacctgagct gagccctgat 1140
gatcctgctg gactgctgga tctgagacag ggcatgtttg cccagctggt ggcccagaat 1200
gtgctgctga ttgatggccc tctgtcctgg tacagcgatc ctggacttgc tggcgttagc 1260
ctgactggcg gcctgagcta caaagaggac accaaagaac tggtggtggc caaggccggc 1320
gtgtactacg tgttctttca gctggaactg cggagagtgg tggccggcga aggatctgga 1380
tctgtgtctc tggctctgca tctgcagcct ctgagatctg ctgctggtgc tgctgctctg 1440
gccctgacag ttgatctgcc tcctgcctct agcgaggcca gaaactccgc ctttggcttc 1500
caaggcagac tgctgcacct gagcgctgga cagagactgg gagtccatct gcacacagaa 1560
gccagagcta gacacgcctg gcagctgaca caaggcgcta cagtgctggg cctgttcaga 1620
gtgacccctg agattccagc cggcctgcca tctcctagat ctgag 1665
<210> 8
<211> 1647
<212> DNA
<213> Artificial sequence
<220>
<221> gene
<222> (1)..(1647)
<223> nucleic acid sequence of SIRPalpha-41 BBL
<400> 8
gaagaggaac tgcaagtgat ccagcctgac aagagcgtgc tggtggctgc tggcgaaaca 60
gccacactga gatgtaccgc cacctctctg atccctgtgg gccctatcca gtggtttaga 120
ggcgctggac ctggcagaga gctgatctac aaccagaaag agggacactt ccccagagtg 180
accaccgtgt ccgacctgac caagcggaac aacatggact tcagcatccg gatcggcaac 240
atcacccctg ccgatgccgg cacctactac tgcgtgaagt tcagaaaggg cagccccgac 300
gacgtcgagt ttaaaagcgg agccggcaca gagctgagcg tgcgggctaa accttctgct 360
cctgtggtgt ctggacctgc cgctagagct acacctcagc acaccgtgtc ttttacctgc 420
gagagccacg gcttcagccc cagagatatc accctgaagt ggttcaagaa cggcaacgag 480
ctgtccgact tccagaccaa cgtggaccct gtgggagaga gcgtgtccta cagcatccac 540
agcacagcca aggtggtgct gacccgggaa gatgtgcact cccaagtgat ttgcgaggtg 600
gcccacgtta ccctgcaagg cgatcctctg agaggcaccg ccaatctgag cgagacaatc 660
cgggtgccac ctacactgga agtgacccag cagcctgtgc gggccgagaa tcaagtgaac 720
gtgacctgcc aagtgcggaa gttctaccct cagagactgc agctgacctg gctggaaaac 780
ggcaatgtgt ccagaaccga gacagccagc accgtgaccg agaacaagga tggcacctac 840
aattggatga gctggctgct cgtgaatgtg tctgcccacc gggacgatgt gaagctgaca 900
tgccaggtgg aacacgatgg ccagcctgcc gtgtctaaga gccacgacct gaaggtgtcc 960
gctcatccca aagagcaggg ctctaatact gccgccgaga acaccggcag caacgagaga 1020
aatatctacg gcgcttgtcc ttgggccgtt tctggcgcta gagcctctcc tggatctgcc 1080
gcttctccca gactgagaga gggacctgag ctgagccctg atgatcctgc tggactgctg 1140
gatctgagac agggcatgtt tgcccagctg gtggcccaga atgtgctgct gattgatggc 1200
cctctgtcct ggtacagcga tcctggactt gctggcgtta gcctgactgg cggcctgagc 1260
tacaaagagg acaccaaaga actggtggtg gccaaggccg gcgtgtacta cgtgttcttt 1320
cagctggaac tgcggagagt ggtggccggc gaaggatctg gatctgtgtc tctggctctg 1380
catctgcagc ctctgagatc tgctgctggt gctgctgctc tggccctgac agttgatctg 1440
cctcctgcct ctagcgaggc cagaaactcc gcctttggct tccaaggcag actgctgcac 1500
ctgagcgctg gacagagact gggagtccat ctgcacacag aagccagagc tagacacgcc 1560
tggcagctga cacaaggcgc tacagtgctg ggcctgttca gagtgacccc tgagattcca 1620
gccggcctgc catctcctag atctgag 1647
<210> 9
<211> 504
<212> PRT
<213> Artificial sequence
<220>
<221> PEPTIDE
<222> (1)..(504)
<223> full-Length amino acid sequence of SIRPalpha
<400> 9
Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys
1 5 10 15
Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu
20 25 30
Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly
35 40 45
Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly
50 55 60
Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr
65 70 75 80
Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu
85 90 95
Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr
100 105 110
Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser
115 120 125
Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
130 135 140
Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
145 150 155 160
Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
165 170 175
Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
180 185 190
Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser
195 200 205
Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
210 215 220
Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
225 230 235 240
Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu
245 250 255
Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
260 265 270
Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
275 280 285
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
290 295 300
Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
305 310 315 320
Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
325 330 335
Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His
340 345 350
Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn
355 360 365
Glu Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val
370 375 380
Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys Lys
385 390 395 400
Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn
405 410 415
Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu
420 425 430
Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn
435 440 445
Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser
450 455 460
Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg
465 470 475 480
Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr
485 490 495
Ala Ser Val Gln Val Pro Arg Lys
500
<210> 10
<211> 1512
<212> DNA
<213> Artificial sequence
<220>
<221> gene
<222> (1)..(1512)
<223> full-length SIRPalpha nucleic acid sequences
<400> 10
atggagcccg ccggcccggc ccccggccgc ctcgggccgc tgctctgcct gctgctcgcc 60
gcgtcctgcg cctggtcagg agtggcgggt gaggaggagc tgcaggtgat tcagcctgac 120
aagtccgtat cagttgcagc tggagagtcg gccattctgc actgcactgt gacctccctg 180
atccctgtgg ggcccatcca gtggttcaga ggagctggac cagcccggga attaatctac 240
aatcaaaaag aaggccactt cccccgggta acaactgttt cagagtccac aaagagagaa 300
aacatggact tttccatcag catcagtaac atcaccccag cagatgccgg cacctactac 360
tgtgtgaagt tccggaaagg gagccctgac acggagttta agtctggagc aggcactgag 420
ctgtctgtgc gtgccaaacc ctctgccccc gtggtatcgg gccctgcggc gagggccaca 480
cctcagcaca cagtgagctt cacctgcgag tcccacggct tctcacccag agacatcacc 540
ctgaaatggt tcaaaaatgg gaatgagctc tcagacttcc agaccaacgt ggaccccgta 600
ggagagagcg tgtcctacag catccacagc acagccaagg tggtgctgac ccgcgaggac 660
gttcactctc aagtcatctg cgaggtggcc cacgtcacct tgcaggggga ccctcttcgt 720
gggactgcca acttgtctga gaccatccga gttccaccca ccttggaggt tactcaacag 780
cccgtgaggg cagagaacca ggtgaatgtc acctgccagg tgaggaagtt ctacccccag 840
agactacagc tgacctggtt ggagaatgga aacgtgtccc ggacagaaac ggcctcaacc 900
gttacagaga acaaggatgg tacctacaac tggatgagct ggctcctggt gaatgtatct 960
gcccacaggg atgatgtgaa gctcacctgc caggtggagc atgacgggca gccagcggtc 1020
agcaaaagcc atgacctgaa ggtctcagcc cacccgaagg agcagggctc aaataccgcc 1080
gctgagaaca ctggatctaa tgaacggaac atctatattg tggtgggtgt ggtgtgcacc 1140
ttgctggtgg ccctactgat ggcggccctc tacctcgtcc gaatcagaca gaagaaagcc 1200
cagggctcca cttcttctac aaggttgcat gagcccgaga agaatgccag agaaataaca 1260
caggacacaa atgatatcac atatgcagac ctgaacctgc ccaaggggaa gaagcctgct 1320
ccccaggctg cggagcccaa caaccacacg gagtatgcca gcattcagac cagcccgcag 1380
cccgcgtcgg aggacaccct cacctatgct gacctggaca tggtccacct caaccggacc 1440
cccaagcagc cggcccccaa gcctgagccg tccttctcag agtacgccag cgtccaggtc 1500
ccgaggaagt ga 1512
<210> 11
<211> 1029
<212> DNA
<213> Artificial sequence
<220>
<221> gene
<222> (1)..(1029)
<223> nucleic acid sequence encoding the extracellular Domain of human SIRP alpha protein
<400> 11
gaggaggagc tgcaggtgat tcagcctgac aagtccgtat cagttgcagc tggagagtcg 60
gccattctgc actgcactgt gacctccctg atccctgtgg ggcccatcca gtggttcaga 120
ggagctggac cagcccggga attaatctac aatcaaaaag aaggccactt cccccgggta 180
acaactgttt cagagtccac aaagagagaa aacatggact tttccatcag catcagtaac 240
atcaccccag cagatgccgg cacctactac tgtgtgaagt tccggaaagg gagccctgac 300
acggagttta agtctggagc aggcactgag ctgtctgtgc gtgccaaacc ctctgccccc 360
gtggtatcgg gccctgcggc gagggccaca cctcagcaca cagtgagctt cacctgcgag 420
tcccacggct tctcacccag agacatcacc ctgaaatggt tcaaaaatgg gaatgagctc 480
tcagacttcc agaccaacgt ggaccccgta ggagagagcg tgtcctacag catccacagc 540
acagccaagg tggtgctgac ccgcgaggac gttcactctc aagtcatctg cgaggtggcc 600
cacgtcacct tgcaggggga ccctcttcgt gggactgcca acttgtctga gaccatccga 660
gttccaccca ccttggaggt tactcaacag cccgtgaggg cagagaacca ggtgaatgtc 720
acctgccagg tgaggaagtt ctacccccag agactacagc tgacctggtt ggagaatgga 780
aacgtgtccc ggacagaaac ggcctcaacc gttacagaga acaaggatgg tacctacaac 840
tggatgagct ggctcctggt gaatgtatct gcccacaggg atgatgtgaa gctcacctgc 900
caggtggagc atgacgggca gccagcggtc agcaaaagcc atgacctgaa ggtctcagcc 960
cacccgaagg agcagggctc aaataccgcc gctgagaaca ctggatctaa tgaacggaac 1020
atctatatt 1029
<210> 12
<211> 254
<212> PRT
<213> Artificial sequence
<220>
<221> PEPTIDE
<222> (1)..(254)
<223> 41BBL full-length amino acid sequence
<400> 12
Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro
1 5 10 15
Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val
20 25 30
Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe
35 40 45
Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser
50 55 60
Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp
65 70 75 80
Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val
85 90 95
Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp
100 105 110
Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu
115 120 125
Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe
130 135 140
Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser
145 150 155 160
Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala
165 170 175
Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala
180 185 190
Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala
195 200 205
Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His
210 215 220
Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val
225 230 235 240
Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
245 250
<210> 13
<211> 765
<212> DNA
<213> Artificial sequence
<220>
<221> gene
<222> (1)..(765)
<223> 41BBL full-length nucleic acid sequence
<400> 13
atggaatacg cctctgacgc ttcactggac cccgaagccc cgtggcctcc cgcgccccgc 60
gctcgcgcct gccgcgtact gccttgggcc ctggtcgcgg ggctgctgct gctgctgctg 120
ctcgctgccg cctgcgccgt cttcctcgcc tgcccctggg ccgtgtccgg ggctcgcgcc 180
tcgcccggct ccgcggccag cccgagactc cgcgagggtc ccgagctttc gcccgacgat 240
cccgccggcc tcttggacct gcggcagggc atgtttgcgc agctggtggc ccaaaatgtt 300
ctgctgatcg atgggcccct gagctggtac agtgacccag gcctggcagg cgtgtccctg 360
acggggggcc tgagctacaa agaggacacg aaggagctgg tggtggccaa ggctggagtc 420
tactatgtct tctttcaact agagctgcgg cgcgtggtgg ccggcgaggg ctcaggctcc 480
gtttcacttg cgctgcacct gcagccactg cgctctgctg ctggggccgc cgccctggct 540
ttgaccgtgg acctgccacc cgcctcctcc gaggctcgga actcggcctt cggtttccag 600
ggccgcttgc tgcacctgag tgccggccag cgcctgggcg tccatcttca cactgaggcc 660
agggcacgcc atgcctggca gcttacccag ggcgccacag tcttgggact cttccgggtg 720
acccccgaaa tcccagccgg actcccttca ccgaggtcgg aataa 765
<210> 14
<211> 615
<212> DNA
<213> Artificial sequence
<220>
<221> gene
<222> (1)..(615)
<223> nucleic acid sequence encoding the extracellular domain of human 41BBL
<400> 14
gcctgcccct gggccgtgtc cggggctcgc gcctcgcccg gctccgcggc cagcccgaga 60
ctccgcgagg gtcccgagct ttcgcccgac gatcccgccg gcctcttgga cctgcggcag 120
ggcatgtttg cgcagctggt ggcccaaaat gttctgctga tcgatgggcc cctgagctgg 180
tacagtgacc caggcctggc aggcgtgtcc ctgacggggg gcctgagcta caaagaggac 240
acgaaggagc tggtggtggc caaggctgga gtctactatg tcttctttca actagagctg 300
cggcgcgtgg tggccggcga gggctcaggc tccgtttcac ttgcgctgca cctgcagcca 360
ctgcgctctg ctgctggggc cgccgccctg gctttgaccg tggacctgcc acccgcctcc 420
tccgaggctc ggaactcggc cttcggtttc cagggccgct tgctgcacct gagtgccggc 480
cagcgcctgg gcgtccatct tcacactgag gccagggcac gccatgcctg gcagcttacc 540
cagggcgcca cagtcttggg actcttccgg gtgacccccg aaatcccagc cggactccct 600
tcaccgaggt cggaa 615
<210> 15
<211> 1749
<212> DNA
<213> Artificial sequence
<220>
<221> gene
<222> (1)..(1749)
<223> cloning sequence of His-tagged SIRPalpha-41 BBL in vector
<400> 15
gaattcccgc cgccaccatg ggctggtcct gcatcattct gtttctggtg gccacagcca 60
ccggcgtgca ctctcaccat catcaccacc atgaagagga actgcaagtg atccagcctg 120
acaagagcgt gctggtggct gctggcgaaa cagccacact gagatgtacc gccacctctc 180
tgatccctgt gggccctatc cagtggttta gaggcgctgg acctggcaga gagctgatct 240
acaaccagaa agagggacac ttccccagag tgaccaccgt gtccgacctg accaagcgga 300
acaacatgga cttcagcatc cggatcggca acatcacccc tgccgatgcc ggcacctact 360
actgcgtgaa gttcagaaag ggcagccccg acgacgtcga gtttaaaagc ggagccggca 420
cagagctgag cgtgcgggct aaaccttctg ctcctgtggt gtctggacct gccgctagag 480
ctacacctca gcacaccgtg tcttttacct gcgagagcca cggcttcagc cccagagata 540
tcaccctgaa gtggttcaag aacggcaacg agctgtccga cttccagacc aacgtggacc 600
ctgtgggaga gagcgtgtcc tacagcatcc acagcacagc caaggtggtg ctgacccggg 660
aagatgtgca ctcccaagtg atttgcgagg tggcccacgt taccctgcaa ggcgatcctc 720
tgagaggcac cgccaatctg agcgagacaa tccgggtgcc acctacactg gaagtgaccc 780
agcagcctgt gcgggccgag aatcaagtga acgtgacctg ccaagtgcgg aagttctacc 840
ctcagagact gcagctgacc tggctggaaa acggcaatgt gtccagaacc gagacagcca 900
gcaccgtgac cgagaacaag gatggcacct acaattggat gagctggctg ctcgtgaatg 960
tgtctgccca ccgggacgat gtgaagctga catgccaggt ggaacacgat ggccagcctg 1020
ccgtgtctaa gagccacgac ctgaaggtgt ccgctcatcc caaagagcag ggctctaata 1080
ctgccgccga gaacaccggc agcaacgaga gaaatatcta cggcgcttgt ccttgggccg 1140
tttctggcgc tagagcctct cctggatctg ccgcttctcc cagactgaga gagggacctg 1200
agctgagccc tgatgatcct gctggactgc tggatctgag acagggcatg tttgcccagc 1260
tggtggccca gaatgtgctg ctgattgatg gccctctgtc ctggtacagc gatcctggac 1320
ttgctggcgt tagcctgact ggcggcctga gctacaaaga ggacaccaaa gaactggtgg 1380
tggccaaggc cggcgtgtac tacgtgttct ttcagctgga actgcggaga gtggtggccg 1440
gcgaaggatc tggatctgtg tctctggctc tgcatctgca gcctctgaga tctgctgctg 1500
gtgctgctgc tctggccctg acagttgatc tgcctcctgc ctctagcgag gccagaaact 1560
ccgcctttgg cttccaaggc agactgctgc acctgagcgc tggacagaga ctgggagtcc 1620
atctgcacac agaagccaga gctagacacg cctggcagct gacacaaggc gctacagtgc 1680
tgggcctgtt cagagtgacc cctgagattc cagccggcct gccatctcct agatctgagt 1740
gataagctt 1749

Claims (24)

1. A sirpa-4-1 BBL fusion protein characterized in that: the SIRP alpha-4-1 BBL fusion protein is shown as SEQ ID NO. 1.
2. A polynucleotide, characterized in that: the polynucleotide is used for encoding the SIRPalpha-4-1 BBL fusion protein of claim 1.
3. A nucleic acid construct characterized in that: the nucleic acid construct comprising the polynucleotide of claim 2, and a regulatory element for directing expression of the polynucleotide in a host cell.
4. The polynucleotide of claim 2 or the nucleic acid construct of claim 3, wherein: the polynucleotide comprises SEQ ID NO. 8.
5. A host cell, characterized in that: the host cell comprises the sirpa-4-1 BBL fusion protein of claim 1, or the polynucleotide or nucleic acid construct of any one of claims 2 to 4.
6. A method for making a sirpa-4-1 BBL fusion protein, comprising: the method comprises the following steps: expressing the polynucleotide or nucleic acid construct of any one of claims 2 to 4 in a host cell.
7. The method of claim 6, wherein: the method comprises the following steps: isolating the fusion protein.
8. The host cell of claim 5 or the method of any one of claims 6 to 7, wherein: the cells are selected from the group consisting of chinese hamster ovary cells, perc.6 cells, and human embryonic kidney cells 293.
9. Use of the sirpa-4-1 BBL fusion protein of claim 1 in the manufacture of a medicament for the treatment of cancer, wherein: a plurality of cells of the cancer express CD47.
10. Use of the sirpa-4-1 BBL fusion protein of claim 1, a polynucleotide encoding said fusion protein, a nucleic acid construct encoding said fusion protein, or a host cell expressing said fusion protein, for the preparation of a medicament for the treatment of cancer, wherein: a plurality of cells of the cancer express CD47.
11. An article of manufacture, characterized by: the article comprises: a packaging material containing an anticancer agent; and the sirpa-4-1 BBL fusion protein of claim 1, a polynucleotide encoding said fusion protein, a nucleic acid construct encoding said fusion protein, or a host cell expressing said fusion protein.
12. An anticancer agent; and the use of the sirpa-4-1 BBL fusion protein of claim 1, a polynucleotide encoding said fusion protein, a nucleic acid construct encoding said fusion protein, or a host cell expressing said fusion protein, in the manufacture of a medicament for the treatment of cancer, wherein: a plurality of cells of the cancer express CD47.
13. Use according to any one of claims 9 to 10 and 12, characterized in that: the cancer is selected from the group consisting of lymphoma, leukemia, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, and squamous cell carcinoma.
14. A method for activating a plurality of T cells, characterized by: the method comprises the following steps: activating the plurality of T cells in vitro in the presence of the sirpa-4-1 BBL fusion protein of claim 1 and a plurality of cancer cells expressing CD47 or exogenous CD 47.
15. A method for activating a plurality of phagocytes, characterized by: the method comprises the following steps: activating the plurality of phagocytes in vitro in the presence of the sirpa-4-1 BBL fusion protein of claim 1 and a plurality of cancer cells expressing CD47 or exogenous CD 47.
16. A method for activating a plurality of immune cells, characterized by: the method comprises the following steps: the sirpa-4-1 BBL fusion protein of claim 1, a polynucleotide encoding said fusion protein, a nucleic acid construct encoding said fusion protein, or a host cell expressing said fusion protein; and activating the plurality of immune cells in vitro in the presence of a plurality of cancer cells expressing CD47 or exogenous CD 47.
17. The use according to any one of claims 9 to 10 and 12, or the method according to claim 16, characterized in that: the cancer is selected from the group consisting of lymphoma, epithelial cancer, and leukemia.
18. The use according to any one of claims 9 to 10 and 12, or the method according to claim 16, characterized in that: the cancer is selected from the group consisting of leukemia and colon cancer.
19. The method of any one of claims 14 to 18, wherein: the activation is performed in the presence of an anticancer agent.
20. The article of manufacture of claim 11, the use of claim 12, or the method of claim 19, wherein: the anti-cancer agent comprises an antibody.
21. The article of manufacture, the use, or the method of claim 20, wherein: the antibody is selected from the group consisting of rituximab, cetuximab, trastuzumab, ibritumomab, alemtuzumab, gemtuzumab, tiimumab, panitumumab, belimumab, bevacizumab, bivalizumab-maytansine, bonafumab, brinzofuzumab, bentuzumab-vitamin, cetuximab, daclizumab, adalimumab, bei Zuoluo mab, cetuximab-pekou, siameb-bogus, darbemumab, ding Tuo mab, ilomumab, ermomab Ma Suoshan antibody, etamomab, gemtuzumab-ozagrimocin, ji Tuo mab, nesuximab, oxbizumab You Tuozhu, oxuzumab, paltuzumab, ramuximab, trastuzumab, tuximab, trastuzumab and ibritumomab.
22. The article of manufacture, the use, or the method of claim 20, wherein: the antibody is selected from the group consisting of rituximab, cetuximab, and alemtuzumab.
23. The method of any one of claims 16 to 22, wherein: the plurality of immune cells comprises T cells.
24. The method of any one of claims 16 to 22, wherein: the plurality of immune cells comprises phagocytes.
CN201880015833.1A 2017-01-05 2018-01-04 SIRP alpha-41 BBL fusion proteins and methods of use thereof Active CN110573522B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201762442469P 2017-01-05 2017-01-05
US62/442,469 2017-01-05
PCT/IL2018/050017 WO2018127919A1 (en) 2017-01-05 2018-01-04 A SIRP1 alpha-41BBL FUSION PROTEIN AND METHODS OF USE THEREOF

Publications (2)

Publication Number Publication Date
CN110573522A CN110573522A (en) 2019-12-13
CN110573522B true CN110573522B (en) 2023-09-19

Family

ID=62790985

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201880015833.1A Active CN110573522B (en) 2017-01-05 2018-01-04 SIRP alpha-41 BBL fusion proteins and methods of use thereof

Country Status (17)

Country Link
US (3) US11130796B2 (en)
EP (2) EP3939993A1 (en)
JP (1) JP7194324B2 (en)
KR (1) KR102581439B1 (en)
CN (1) CN110573522B (en)
AU (1) AU2018205890B2 (en)
CA (1) CA3047708A1 (en)
DK (1) DK3565828T3 (en)
ES (1) ES2916217T3 (en)
HR (1) HRP20220230T1 (en)
HU (1) HUE057326T2 (en)
IL (1) IL267862B1 (en)
PL (1) PL3565828T3 (en)
PT (1) PT3565828T (en)
RU (1) RU2769769C2 (en)
SI (1) SI3565828T1 (en)
WO (1) WO2018127919A1 (en)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11299530B2 (en) 2017-01-05 2022-04-12 Kahr Medical Ltd. SIRP alpha-CD70 fusion protein and methods of use thereof
WO2018127916A1 (en) 2017-01-05 2018-07-12 Kahr Medical Ltd. A pd1-cd70 fusion protein and methods of use thereof
LT3565579T (en) 2017-01-05 2023-09-11 Kahr Medical Ltd. A pd1-41bbl fusion protein and methods of use thereof
CA3047708A1 (en) 2017-01-05 2018-07-12 Kahr Medical Ltd. A sirpalpha-41bbl fusion protein and methods of use thereof
WO2020012486A1 (en) * 2018-07-11 2020-01-16 Kahr Medical Ltd. SIRPalpha-4-1BBL VARIANT FUSION PROTEIN AND METHODS OF USE THEREOF
KR20220044284A (en) * 2019-07-11 2022-04-07 카 메디컬 리미티드 Heterodimer and method of use thereof
WO2021137230A1 (en) 2019-12-31 2021-07-08 Kahr Medical Ltd. Methods of culturing t cells and uses of same
US20230048719A1 (en) 2019-12-31 2023-02-16 Kahr Medical Ltd. Methods of culturing t cells with a 4-1bbl fusion polypeptide and uses of same
KR20220138909A (en) * 2021-04-06 2022-10-14 주식회사 이뮤노로지컬디자이닝랩 Chimeric antigen receptor specifically binding to CD47 and use thereof
WO2022215920A1 (en) * 2021-04-06 2022-10-13 주식회사 이뮤노로지컬디자이닝랩 Transformed antigen-specific professional antigen-presenting cells comprising chimeric antigen receptor (car) and use thereof
TW202400650A (en) 2022-05-11 2024-01-01 日商第一三共股份有限公司 Combination of an antibody and cd47 inhibitor

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011109789A2 (en) * 2010-03-05 2011-09-09 The Johns Hopkins University Compositions and methods for targeted immunomodulatory antibodies and fusion proteins

Family Cites Families (108)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4475196A (en) 1981-03-06 1984-10-02 Zor Clair G Instrument for locating faults in aircraft passenger reading light and attendant call control system
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4486194A (en) 1983-06-08 1984-12-04 James Ferrara Therapeutic device for administering medicaments through the skin
US4596556A (en) 1985-03-25 1986-06-24 Bioject, Inc. Hypodermic injection apparatus
US5374548A (en) 1986-05-02 1994-12-20 Genentech, Inc. Methods and compositions for the attachment of proteins to liposomes using a glycophospholipid anchor
MX9203291A (en) 1985-06-26 1992-08-01 Liposome Co Inc LIPOSOMAS COUPLING METHOD.
WO1987005330A1 (en) 1986-03-07 1987-09-11 Michel Louis Eugene Bergh Method for enhancing glycoprotein stability
DE3751873T2 (en) 1986-04-09 1997-02-13 Genzyme Corp Genetically transformed animals that secrete a desired protein in milk
US4941880A (en) 1987-06-19 1990-07-17 Bioject, Inc. Pre-filled ampule and non-invasive hypodermic injection device assembly
US4790824A (en) 1987-06-19 1988-12-13 Bioject, Inc. Non-invasive hypodermic injection device
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
US5108921A (en) 1989-04-03 1992-04-28 Purdue Research Foundation Method for enhanced transmembrane transport of exogenous molecules
US5464764A (en) 1989-08-22 1995-11-07 University Of Utah Research Foundation Positive-negative selection methods and vectors
US5064413A (en) 1989-11-09 1991-11-12 Bioject, Inc. Needleless hypodermic injection device
US5312335A (en) 1989-11-09 1994-05-17 Bioject Inc. Needleless hypodermic injection device
US5287386A (en) 1991-03-27 1994-02-15 Thinking Machines Corporation Differential driver/receiver circuit
US5541104A (en) 1991-05-23 1996-07-30 Ludwig Institute For Cancer Research Monoclonal antibodies which bind to tumor rejection antigen precursor mage-1
US5342774A (en) 1991-05-23 1994-08-30 Ludwig Institute For Cancer Research Nucleotide sequence encoding the tumor rejection antigen precursor, MAGE-1
US6235525B1 (en) 1991-05-23 2001-05-22 Ludwig Institute For Cancer Research Isolated nucleic acid molecules coding for tumor rejection antigen precursor MAGE-3 and uses thereof
US5383851A (en) 1992-07-24 1995-01-24 Bioject Inc. Needleless hypodermic injection device
JP3608788B2 (en) 1992-08-31 2005-01-12 ルドヴィグ・インスティテュート・フォー・キャンサー・リサーチ Isolated nonapeptides derived from the MAGE-3 gene and presented by HLA-A1 and their uses
US6222012B1 (en) 1992-08-31 2001-04-24 Ludwig Institute For Cancer Research Isolated nonapeptides presented by HLA molecules, and uses thereof
US5674704A (en) 1993-05-07 1997-10-07 Immunex Corporation Cytokine designated 4-IBB ligand
DE69524264T2 (en) 1994-03-01 2002-07-18 Ludwig Inst Cancer Res DETERMINATION OF CANCER-SPECIFIC TEXTURES BY GENE EXPRESSION OF A MEMBER OF THE MELANOMARIAN GROUP, MAGE
US5541110A (en) 1994-05-17 1996-07-30 Bristol-Myers Squibb Cloning and expression of a gene encoding bryodin 1 from Bryonia dioica
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US6046048A (en) 1996-01-09 2000-04-04 Genetech, Inc. Apo-2 ligand
US6291430B1 (en) 1997-09-12 2001-09-18 Ludwig Institute For Cancer Research Mage-3 peptides presented by HLA class II molecules
US6740739B1 (en) 1998-01-15 2004-05-25 Genentech, Inc. Substitutional variants of APO-2 ligand
AU1811801A (en) 1999-12-03 2001-06-12 Dendreon Corporation Cryopreservation of antigen-loaded dendritic cells and their precursors in serum-free media
WO2001049318A1 (en) 2000-01-03 2001-07-12 Tr Associates, L.L.C. Novel chimeric proteins and methods for using the same
WO2001057251A2 (en) 2000-02-04 2001-08-09 Aeomica, Inc. Methods and apparatus for predicting, confirming, and displaying functional information derived from genomic sequence
US6436703B1 (en) 2000-03-31 2002-08-20 Hyseq, Inc. Nucleic acids and polypeptides
JP4446036B2 (en) 2001-11-27 2010-04-07 ウーセーベー ファルマ ソシエテ アノニム Pharmaceutical composition for the treatment of epithelial induced cancer
US7772188B2 (en) * 2003-01-28 2010-08-10 Ironwood Pharmaceuticals, Inc. Methods and compositions for the treatment of gastrointestinal disorders
KR20050082389A (en) 2004-02-18 2005-08-23 메덱스젠 주식회사 Pharmaceutical composition for treatment of transplantation rejection comprising concatameric immunoadhesin
US7142018B2 (en) 2004-06-08 2006-11-28 Transmeta Corporation Circuits and methods for detecting and assisting wire transitions
JP5948627B2 (en) 2004-10-29 2016-07-20 レイショファーム ゲーエムベーハー Fibroblast growth factor (FGF) remodeling and carbohydrate pegylation
WO2006050262A2 (en) 2004-11-01 2006-05-11 The Regents Of The University Of California Compositions and methods for modification of biomolecules
US7279925B1 (en) 2005-03-10 2007-10-09 Cypress Semiconductor Corp. Capacitive feedforward circuit, system, and method to reduce buffer propagation delay
JP2006345852A (en) 2005-06-16 2006-12-28 Virxsys Corp Antibody conjugate
EP1787512A1 (en) 2005-11-09 2007-05-23 Paul-Ehrlich-Institut Bundesamt für Sera und Impfstoffe Method for the cryopreservation of human blood
KR101855381B1 (en) 2008-04-09 2018-05-09 제넨테크, 인크. Novel compositions and methods for the treatment of immune related diseases
AU2009269141B2 (en) 2008-06-30 2013-04-04 University Of Pennsylvania Fn14/TRAIL fusion proteins
BRPI0917891A2 (en) 2008-08-25 2015-11-24 Amplimmune Inc pd-1 antagonists and methods of using them
JP2012512640A (en) 2008-12-19 2012-06-07 ノバルティス アーゲー Soluble polypeptides for use in the treatment of autoimmune and inflammatory disorders
FI2995315T3 (en) 2009-05-15 2024-03-18 Univ Health Network Compositions and methods for treating hematologic cancers targeting the sirp alpha- cd47 interaction
EP2468850A4 (en) 2009-08-19 2013-02-20 Takara Bio Inc Cell preservation method
EP2621514B1 (en) 2010-09-28 2016-09-21 KAHR Medical (2005) Ltd Compositions and methods for treatment of hematological malignancies
US8675439B2 (en) 2011-10-12 2014-03-18 Taiwan Semiconductor Manufacturing Co., Ltd. Bit line voltage bias for low power memory design
US9926364B2 (en) 2011-11-03 2018-03-27 Argen-X N.V. Chimeric human-llama antigens and methods of use
DK2804617T3 (en) 2012-01-17 2020-08-10 Univ Leland Stanford Junior High affinity sirp alpha reagents
US20140370012A1 (en) 2012-01-27 2014-12-18 Gliknik Inc. Fusion proteins comprising igg2 hinge domains
CN104245726A (en) 2012-03-29 2014-12-24 奥瑞基尼探索技术有限公司 Immunomodulating cyclic compounds from the bc loop of human PD1
EP3489254B9 (en) 2012-04-30 2022-12-21 Biocon Limited Targeted/immunomodulatory fusion proteins and methods for making same
EP2730588A1 (en) 2012-11-12 2014-05-14 Intelectys Antibodies and fragments thereof raised against the alpha-3 domain of HLA-G protein, methods and means for their preparation, and uses thereof
CN104968364A (en) 2012-12-03 2015-10-07 百时美施贵宝公司 Enhancing anti-cancer activity of immunomodulatory Fc fusion proteins
EP2941438A1 (en) 2013-01-01 2015-11-11 Kahr Medical Ltd. Stable form of signal converting protein fusion proteins, and methods of use and preparation thereof
US9873747B2 (en) * 2013-01-31 2018-01-23 Thomas Jefferson University Fusion proteins that facilitate cancer cell destruction
ES2686968T3 (en) 2013-05-06 2018-10-23 China Pharmaceutical University Bifunctional fusion proteins to inhibit angiogenesis in the tumor microenvironment and to activate adaptive immune responses and their genes and uses
WO2015148416A1 (en) * 2014-03-24 2015-10-01 Deqiang Jing Novel recombinant bi-functional fusion proteins, preparation and use thereof
EP3177640B1 (en) 2014-08-08 2020-05-06 The Board of Trustees of the Leland Stanford Junior University High affinity pd-1 agents and methods of use
WO2016023001A1 (en) 2014-08-08 2016-02-11 The Board Of Trustees Of The Leland Stanford Junior University Multispecific high affinity pd-1 agents and methods of use
EP3643727A1 (en) * 2014-08-15 2020-04-29 Merck Patent GmbH Sirp-alpha immunoglobulin fusion proteins
EP3012271A1 (en) 2014-10-24 2016-04-27 Effimune Method and compositions for inducing differentiation of myeloid derived suppressor cell to treat cancer and infectious diseases
MX2017006250A (en) 2014-11-14 2017-11-17 Hoffmann La Roche Antigen binding molecules comprising a tnf family ligand trimer.
CN104403004B (en) 2014-11-24 2017-10-13 苏州丁孚靶点生物技术有限公司 The preparation and use of antibody interferon heterodimer
RS62870B1 (en) 2014-12-05 2022-02-28 Memorial Sloan Kettering Cancer Center Chimeric antigen receptors targeting g-protein coupled receptor and uses thereof
KR20180003538A (en) 2015-03-03 2018-01-09 카 메디컬 리미티드 Dual signaling protein (DSP) fusion proteins and their use in the treatment of diseases
EP3277305B1 (en) 2015-03-31 2021-07-21 F. Hoffmann-La Roche AG Antigen binding molecules comprising a trimeric tnf family ligand
CN106146670B (en) * 2015-04-24 2019-01-15 宜明昂科生物医药技术（上海）有限公司 A kind of new recombination double functions fusion protein and its preparation and application
WO2016187226A1 (en) * 2015-05-18 2016-11-24 Ab Initio Biotherapeutics, Inc. Sirp polypeptide compositions and methods of use
WO2017012770A1 (en) 2015-07-23 2017-01-26 Ratiopharm Gmbh Acid addition salts of filgotinib
GEP20227419B (en) 2015-07-30 2022-10-10 Macrogenics Inc Pd-1-binding molecules and methods of use thereof
BR102016018074A2 (en) 2015-08-07 2021-11-16 ALX Oncology Inc. SIRP-ALFA VARIANT CONSTRUCTION, ITS METHOD OF PREPARATION AND USES, NUCLEIC ACID MOLECULE, VECTOR, HOST CELL, AND PHARMACEUTICAL COMPOSITION
PT3331902T (en) 2015-08-07 2021-07-26 Alx Oncology Inc Constructs having a sirp-alpha domain or variant thereof
BR112018006578A2 (en) 2015-10-01 2018-12-26 Heat Biologics, Inc. compositions and methods for joining type i and type ii extracellular domains as heterologous chimeric proteins
CA3002602C (en) 2015-10-23 2021-11-02 Apogenix Ag Single-chain cd27-receptor agonist proteins
WO2017152132A1 (en) 2016-03-04 2017-09-08 The Board Of Trustees Of The Leland Stanford Junior University Methods of identifying and treating immune checkpoint inhibitor-responsive neoplasms
CN109715808A (en) 2016-04-15 2019-05-03 诺华股份有限公司 Composition and method for selective protein expression
MA44993A (en) 2016-05-13 2019-03-20 Medimmune Llc CD40L-FC FUSION POLYPEPTIDES AND ASSOCIATED PROCEDURES FOR USE
EP3243832A1 (en) 2016-05-13 2017-11-15 F. Hoffmann-La Roche AG Antigen binding molecules comprising a tnf family ligand trimer and pd1 binding moiety
BR112018074847A2 (en) 2016-06-03 2019-03-26 Invectys anti hla-g specific antibodies
CN107586342A (en) 2016-07-08 2018-01-16 生命序有限公司 Recombinant immune checkpoint acceptor and its application
CN106267188A (en) 2016-08-15 2017-01-04 深圳大学 The novel antibodies of small molecule immune agonist coupling PD 1 antibody and the application in antitumor thereof
CN107868791B (en) 2016-09-26 2021-11-23 阿思科力(苏州)生物科技有限公司 Preparation method and application of reinforced Slit2CAR-T and CAR-NK cells
US10654929B2 (en) 2016-11-02 2020-05-19 Jounce Therapeutics, Inc. Antibodies to PD-1 and uses thereof
TW201829463A (en) 2016-11-18 2018-08-16 瑞士商赫孚孟拉羅股份公司 Anti-hla-g antibodies and use thereof
EP3554542A1 (en) 2016-12-19 2019-10-23 H. Hoffnabb-La Roche Ag Combination therapy with targeted 4-1bb (cd137) agonists
CA3047708A1 (en) 2017-01-05 2018-07-12 Kahr Medical Ltd. A sirpalpha-41bbl fusion protein and methods of use thereof
US11299530B2 (en) 2017-01-05 2022-04-12 Kahr Medical Ltd. SIRP alpha-CD70 fusion protein and methods of use thereof
WO2018127916A1 (en) 2017-01-05 2018-07-12 Kahr Medical Ltd. A pd1-cd70 fusion protein and methods of use thereof
LT3565579T (en) 2017-01-05 2023-09-11 Kahr Medical Ltd. A pd1-41bbl fusion protein and methods of use thereof
CN107857819A (en) 2017-07-03 2018-03-30 江苏西迪尔生物技术有限公司 Multi-functional fusion protein and its application
EP3703746A1 (en) 2017-11-01 2020-09-09 F. Hoffmann-La Roche AG Novel tnf family ligand trimer-containing antigen binding molecules
WO2020012486A1 (en) 2018-07-11 2020-01-16 Kahr Medical Ltd. SIRPalpha-4-1BBL VARIANT FUSION PROTEIN AND METHODS OF USE THEREOF
JP2021531265A (en) 2018-07-11 2021-11-18 カール メディカル リミテッド PD1-4-1BBL variant fusion protein and its usage
WO2020023268A1 (en) 2018-07-24 2020-01-30 Amgen Inc. Combination of lilrb1/2 pathway inhibitors and pd-1 pathway inhibitors
CA3125748A1 (en) 2019-01-07 2020-07-16 Thomas Jefferson University Multi-functional fusion proteins and uses thereof
TWI760751B (en) 2019-05-29 2022-04-11 美商美國禮來大藥廠 Tigit and pd-1/tigit-binding molecules
CN110128550B (en) 2019-05-30 2023-01-13 南京惟亚德生物医药有限公司 Novel replicative oncolytic adenovirus capable of simultaneously blocking immune check points PD-L1 and TIGIT and application
KR20220044284A (en) 2019-07-11 2022-04-07 카 메디컬 리미티드 Heterodimer and method of use thereof
US20240076346A1 (en) 2021-01-13 2024-03-07 Kahr Medical Ltd. Type i membrane proteins heterodimers and methods of use thereof
WO2023119295A1 (en) 2021-12-23 2023-06-29 Kahr Medical Ltd. Lilrb polypeptides and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011109789A2 (en) * 2010-03-05 2011-09-09 The Johns Hopkins University Compositions and methods for targeted immunomodulatory antibodies and fusion proteins

Also Published As

Publication number Publication date
EP3565828A4 (en) 2020-11-11
DK3565828T3 (en) 2022-02-21
JP7194324B2 (en) 2022-12-22
US11130796B2 (en) 2021-09-28
WO2018127919A1 (en) 2018-07-12
RU2019124676A (en) 2021-02-06
SI3565828T1 (en) 2022-04-29
WO2018127919A9 (en) 2018-12-13
IL267862A (en) 2019-09-26
AU2018205890A1 (en) 2019-08-08
HRP20220230T1 (en) 2022-04-29
US20240109952A1 (en) 2024-04-04
EP3565828B8 (en) 2022-07-27
US20190352372A1 (en) 2019-11-21
RU2019124676A3 (en) 2021-04-09
IL267862B1 (en) 2024-01-01
PT3565828T (en) 2022-02-08
JP2020505010A (en) 2020-02-20
CA3047708A1 (en) 2018-07-12
RU2769769C2 (en) 2022-04-05
ES2916217T3 (en) 2022-06-29
EP3565828A1 (en) 2019-11-13
US20210371500A1 (en) 2021-12-02
PL3565828T3 (en) 2022-04-04
US11897937B2 (en) 2024-02-13
EP3565828B1 (en) 2021-11-24
AU2018205890B2 (en) 2021-09-02
KR20190122660A (en) 2019-10-30
EP3939993A1 (en) 2022-01-19
KR102581439B1 (en) 2023-09-21
HUE057326T2 (en) 2022-04-28
CN110573522A (en) 2019-12-13

Similar Documents

Publication Publication Date Title
CN110573522B (en) SIRP alpha-41 BBL fusion proteins and methods of use thereof
CN110536693B (en) PD1-41BBL fusion proteins and methods of using the same
EP3820887A1 (en) Pd1-4-1bbl variant fusion protein and methods of use thereof
US20210214417A1 (en) SIRPalpha-4-1BBL VARIANT FUSION PROTEIN AND METHODS OF USE THEREOF
WO2023119295A1 (en) Lilrb polypeptides and uses thereof
RU2815515C2 (en) Embodiments of sirp-alpha-4-1bbl fusion protein and methods of use thereof
CN116981686A (en) Type I membrane protein heterodimers and methods of use thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant